Methods and Compositions for Unsilencing Imprinted Genes

ABSTRACT

The present invention provides methods and compositions for inducing expression of Ube3a in a cell by contacting the cell with a topoisomerase inhibitor. Particular embodiments include a method of treating a genomic imprinting disorder, such as Angelman syndrome, in a subject by administering to the subject an effective amount of a topoisomerase inhibitor.

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. §119(e), of U.S.Provisional Application Ser. No. 61/412,638, filed Nov. 11, 2010, theentire contents of which are incorporated by reference herein.

STATEMENT OF GOVERNMENT SUPPORT

Aspects of this invention were funded under Contract No.W81XWH-10-1-0710 from the Department of Defense. The U.S. Government hascertain rights in this invention.

BACKGROUND OF THE INVENTION

Angelman syndrome is an autism spectrum disorder for which no effectivetreatment currently exists. Individuals with Angelman syndrome exhibitoutwardly normal development during the first year of life, but thendevelop severe intellectual disabilities, seizures, EEG abnormalities,gait disturbances, disrupted sleep patterns, and profound languageimpairments (Zori et al., 1992; Lossie et al., 2001; Clayton-Smith andLaan, 2003; Williams, 2005; Dan, 2008; Pelc et al., 2008; Dan, 2009).These deficits are caused by maternal deletions or mutations of a singlegene, the E3 ubiquitin ligase Ube3a, and can be modeled inUbe3a-deficient mice. Because the paternal allele of Ube3a is silencedin most neurons through epigenetic imprinting, lost function of thematernal allele eliminates Ube3a protein expression in neurons (FIG. 1).With a prevalence of 1:15,000 (Steffenburg et al., 1996; Dan, 2008) andan average cost of care of >$150,000/year per individual across a fulllifespan (Angelman Syndrome Foundation), the health care costs ofAngelman syndrome are immense, quite aside from the human cost.

Genetic engineering approaches were used to rescue the neurologicaldeficits in Angelman syndrome model mice (van Woerden et al., 2007), butthis approach relies on knocking out genes, making it impractical inhumans. A pharmacological approach offers a more viable alternative.Only one clinical trial, which tested the effects of the methyl donorsbetaine and folic acid, has been performed in humans (Arn et al., 1998;Bacino et al., 2003), and it has not proved successful to date.

The present invention provides methods and compositions for unsilencingimprinted genes (e.g., the paternal allele of Ube3a silenced throughepigenetic imprinting), thereby providing methods of treatment ofgenomic imprinting disorders, such as Angelman syndrome.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of inducingexpression of Ube3a in a cell, comprising contacting the cell with aneffective amount of a topoisomerase inhibitor, thereby inducingexpression of Ube3a in the cell.

An additional aspect of the present invention is a method of treating agenomic imprinting disorder in a subject, comprising administering tothe subject an effective amount of a topoisomerase inhibitor, therebytreating the genomic imprinting disorder in the subject.

Further aspects of this invention include a method of treating adisorder associated with an epigenetic modification in a subject,comprising administering to the subject an effective amount of atopoisomerase inhibitor, thereby treating the disorder associated withthe epigenetic modification in the subject.

DESCRIPTION OF THE FIGURES

FIG. 1. Schematic of how Angelman syndrome can be treated by unsilencingthe intact paternal Ube3a allele.

FIG. 2. Schematic of drug discovery approach to identify Angelmansyndrome therapeutics. Unsilencing of the paternal Ube3a allele isassessed by a fluorescence-based assay in a 384-well format. 7 DIVcultures are treated in quadruplicate with 10 μM small molecules or 0.2%DMSO (vehicle) using fluid handling robotics. At 72 hr post-drug or DMSOtreatments, the YFP signal is amplified by using an anti-GFP antibody(which also recognizes YFP) and Ube3a-YFP fluorescence is assessedin >1200 individual neurons/well using the BD Pathway 855.

FIGS. 3A-D. High-content imaging of Ube3a-YFP fluorescence candistinguish between active and silenced Ube3a alleles in corticalneurons in vitro. Cortical neurons are shown at 7 DIV from miceexpressing the Ube3a-YFP fusion protein on the maternal (Ube3a^(Ey/+))or paternal (Ube3a^(+/Ey)) allele. High-content 4×4 montage images show(A) Ube3a-YFP and (B) nuclei stained with DAPI. The insets shown in Aare zoomed and shown in C. YFP fluorescence intensity was quantifiedfrom signals in individual cells after a background subtraction and bysegmenting the cell nuclei area. (D) The mean intensity in all cells inthe montaged fields in quadruplicate wells (>1500 cells total/datapoint) was determined over 10 days in culture for the genotypes(wildtype has no Ube3a-YFP) and expressed as percent paternal Ube3a-YFP.Maternal Ube3a-YFP fluorescence increases after the first few DIV;however, paternal expression is silenced.

FIGS. 4A-D. Screening identifies that the topoisomerase inhibitoririnotecan unsilences paternal Ube3a in vitro. (A) Paternal Ube3a-YFP issilenced (not expressed) in cultured neurons treated with vehicle (0.2%DMSO). (B) Treating cultures for 72 hr with 10 μM irinotecan turns onpaternal Ube3a-YFP. This has been independently reproduced in n=10experiments, each experiment run on separate days in quadruplicate.(C-D) Neuron density and health is similar in vehicle- and drug-treatedcells as evidenced by counterstaining with the nuclear marker DAPI.

FIGS. 5A-B. Additional assays confirm that irinotecan unsilences thepaternal Ube3a-YFP allele. Irinotecan (10 μM for 72 hrs) upregulated thepaternal Ube3a-YFP allele as measured by (A) mRNA levels in quantitativeRT-PCR experiments and by (B) protein levels assessed by Western blots.V1-V3=samples from separate vehicle-treated cultures; D1-D3=samples fromseparate drug-treated cultures. *p<0.05, unpaired t-test.

FIG. 6. Irinotecan can unsilence the paternal Ube3a allele in neuronsfrom Angelman syndrome model mice. Irinotecan (10 μM) significantlyupregulated Ube3a protein levels in neurons that were cultured fromUbe3a^(m−/p+) mice for 72 hrs, with levels reaching ˜50% of controllevels (in Ube3a^(m+/p+) mice). V=vehicle-treated; D=drug-treated.*p<0.01, unpaired t-test.

FIG. 7. Concentration-response curves demonstrating the relativepotencies for irinotecan and topotecan for unsilencing paternalUbe3a-YFP. These concentration-response curves demonstrate thatirinotecan and topotecan have a similar E_(max), but topotecan is ˜20times more potent than irinotecan. Irinotecan EC₅₀=996±10 nM; TopotecanEC₅₀=55±1.2 nM. Quantified data are mean±SEM from wells treated for 72hr (>1200 cells total/culture well; n=4 for each data point).

FIGS. 8A-F. Irinotecan and topotecan can unsilence paternal Ube3a invivo. (A) Paternal Ube3a-YFP is silenced (not expressed) in spinalneurons. Intrathecal injection of (B) irinotecan or (C) topotecan intolive mice unsilenced the paternal Ube3a-YFP allele in spinal neurons.These results have been reproduced in n>4 mice. (D-F) Neuron health issimilar in vehicle- and drug-treated mice as evidenced bycounterstaining with the pan-neuronal marker NeuN. Scale bar=100 μm.

FIGS. 9A-C. One week of topotecan treatment is sufficient to unsilencepaternal Ube3a throughout the brain. (A₁) Coronal section showing thatintracerebroventricular osmotic minipump infusions of vehicle do notunsilence paternal Ube3a-YFP. Boxes highlight regions of the (A₂)neocortex and (A₃) hippocampus, at higher magnifications. (B₁) Topotecanfor one week unsilences paternal Ube3a-YFP throughout the brain. Highermagnifications demonstrate paternal Ube3a-YFP unsilencing in (B₂)neocortex and (B₃) hippocampus. (C) Control labeling of maternalUbe3a-YFP. In this coronal section some of the structures shown in (A₁)and (B₁) are not visible, such as the hippocampus.

FIGS. 10A-D. Topotecan-induced unsilencing of paternal Ube3a islong-lasting. (A) Two weeks of daily i.t. injections of topotecan (100nmol/day on 10 of 14 days) unsilences paternal Ube3a-YFP in a subset ofneurons (compare to more complete paternal unsilencing with higher drugconcentrations in FIG. 9C). (B) 14 days after cessation of topotecaninjections, unsilencing of paternal Ube3a persists, suggesting that theloss of imprinting may be permanent. (C-D) Counterstaining of Ube3a-YFP(green) with the pan-neuronal marker NeuN (blue). Scale bar=100 μm.These experiments were performed in mice expressing Ube3a-YFP on thepaternal, but not maternal, allele.

FIG. 11. Ube3a-deficient mice exhibit deficits in water-maze reversallearning. Significant effects of genotype were observed in reversallearning [main effect of genotype, F(1,19)=7.47, p=0.0132; genotype Xtarget location interaction, F(3,57)=6.29, p=0.0009]. Duringacquisition, within-genotype repeated measures ANOVAs showed targetselectivity in both groups [wildtype, F(3,27)=19.23, p<0.0001; m−/p+,F(3,30)=20.62, p<0.0001]. However, only the wildtype group demonstrateda significant preference for the new platform location [F(3,27)=40.06,p<0.0001]. *p<0.05

FIG. 12. Topoisomerase I RNAi-mediated knockdown increases Ube3a proteinexpression. Western blots were used in preliminary studies to quantifyprotein levels of Ube3a in cultured neurons after 5 days ofRNAi-treatment.

FIGS. 13A-G. A small-molecule screen identifies a topoisomeraseinhibitor that unsilences the paternal allele of Ube3a in neurons. (A)High-content screen flowchart. E15.5 cortical neurons with a paternallyinherited Ube3a-YFP allele were cultured in 384-well plates and treatedwith small molecules from DIV7-DIV10. Active compounds that unsilencethe paternal Ube3a-YFP allele were detected with antibody-enhancedfluorescence and high-content imaging. (B) High-content imaging of DIV7neurons that inherited Ube3a-YFP maternally (m^(YFP)/p⁺) or paternally(m⁺/p^(YFP)). Nuclei were stained with DAPI. Scale bar=50 μm. (C)Mean±s.e.m. levels of UBE3A-YFP fluorescence in neurons cultured frommaternal Ube3a-YFP(m^(YFP)/p⁺) or wild-type (m⁺/p⁺) mice, normalized tolevels in age-matched neurons cultured from paternal Ube3a-YFP mice(m⁺/p^(YFP)). Two-way ANOVA, F_((1,58))=1492.52, P<0.001 for genotype;F_((9,58))=97.72, P<0.001 for duration; F_((9,58))=97.72, P<0.001 forgenotype-duration interactions; Bonferroni post hoc test aftersignificant main effects examined comparisons between maternal andpaternal Ube3a-YFP mice from DIV4 to DIV10, *P<0.001; n=2-6 culturewells/day. (D) Pie chart depicting categories of the 2,306 screenedcompounds and graph summarizing presumptive UBE3A-YFP in arbitraryfluorescence units (A.F.U.) after small molecule treatments. Smallmolecules that were subsequently found to be autofluorescent (FIG. 17)are depicted in gray. The initial screen identified one active compound,irinotecan. (E) High magnification view of wells treated with vehicle(0.2% DMSO) or 10 μM irinotecan for 72 hr. Neuron density and health issimilar in vehicle- and irinotecan-treated cells as evidenced bycounterstaining with the nuclear marker DAPI. Scale bar=100 μm. (F)Western blot showing UBE3A-YFP protein levels were increased withirinotecan (10 μM for 72 h). *P<0.05; two-tailed t-test, n=3/group. (G)Western blot to detect UBE3A protein in cultures taken from wild-type(m⁺/p⁺) or maternally Ube3a-deficient (m⁻/p⁺) mice treated with vehicleor irinotecan (10 μM for 72 h). One-way ANOVA, F_((2,20))=30.47,P<0.001; Bonferroni post hoc test after significant main effect,*P<0.001; n=7-8/group. All data are presented as means±s.e.m.

FIGS. 14A-I. Topotecan unsilences the paternal allele of Ube3a and theunsilenced protein is catalytically active. (A) Dose-response curves forunsilencing the paternal Ube3a-YFP allele. Inactive=lactam Ering-camptothecin. n=4/data point. (B) UBE3A-YFP levels in neurons fromUbe3a^(m+/pYFP) mice increase with duration of topotecan (300 nM) oririnotecan (1 μM) treatment. For topotecan treatment, one-way ANOVA,F_((5,32))=47.73, P<0.001; Bonferroni post hoc test after significantmain effect, *P<0.01 compared to day zero; n=4-7/group. For irinotecantreatment, one-way ANOVA, F_((5,30))=12.17, P<0.001; Bonferroni post hoctest after significant main effect, *P<0.05 compared to day zero;n=4-8/group. A.F.U.=arbitrary fluorescence units. (C) Western blots andquantification of UBE3A and the loading control actin demonstrating thattopotecan restores UBE3A levels in neurons from maternal Ube3a-deficientmice (m⁻/p⁺) to wild-type (m⁺/p⁺) levels. One-way ANOVA, F_((2,9))=8.28,P<0.01; Bonferroni post hoc test after significant main effect, *P<0.05;n=4/group. (D) Quantification of unbound TOP1 and representative Westernblots. Note the decrease of unbound TOPI after topotecan treatment inmaternal Ube3a-deficient (m⁻/p⁺) neurons compared to vehicle-treatedneurons (m⁻/p⁺ or m⁺/p⁺). β-tubulin was used as a loading control.One-way ANOVA, F_((2,6))=17.88, P<0.005; Bonferroni post hoc test aftersignificant main effect, *P<0.05; n=3/group. (E) Western blot fromvehicle- and topotecan-treated neurons from wild-type (m⁺/p⁺) andmaternal Ube3a-deficient (m⁻/p⁺) mice. The higher molecular weight formrepresenting ubiquitin-bound UBE3A is lost by treatment with thereducing agent DTT, which disrupts the UBE3A-thioester-ubiquitin bond.(F) Western blots examining UBE3A ubiquitin-thioester formationfollowing immunoprecipitation with an anti-UBE3A antibody and in vitroubiquitination in the presence or absence of the ubiquitin conjugatingenzyme (E2), UBCH7. Note that UBE3A from wild-type (m⁺/p⁺) andtopotecan-treated maternal Ube3a-deficient (m⁻/p⁺) neurons undergo amobility shift in the presence of UBCH7 that is lost by addition of thereducing agent, DTT. All data are presented as means±s.e.m. (G)Schematic demonstrating location of 4 primer sets used to probe mRNAexpression shown in (H). (H) Normalized mRNA levels in culturedUbe3a^(m−/p+) neurons following vehicle or 300 nM topotecan. Expressionis given as ratio of expression in drug treated cells to vehicle treatedcells, normalized to the housekeeping gene RPL22. *P<0.05 compared to 0hr, Kruskal-Wallis one-way ANOVA followed by post hoc tests, n=4-5cultures/data point. (I) Schematic summarizing methylation status of theSnrpn promoter region on the maternal and paternal chromosome followingtreatment with vehicle or 300 nM topotecan (see complete primer 1 dataset in FIG. 27). Average methylation status is indicated using agrayscale.

FIGS. 15A-H. Topotecan enduringly unsilences the paternal allele ofUbe3a in vivo. (A) Schematic depicts unilateral delivery of topotecan(i.c.v.) using a mini-osmotic pump into the lateral ventricle ofUbe3a^(m+/pYFP) mice in vivo. Two weeks of topotecan infusion (3.74μg/h) unsilenced the paternal Ube3a-YFP allele in the hippocampus of theinfused hemisphere near the site of drug delivery, while only modestlyunsilencing Ube3a-YFP in the contralateral (non-infused) hemisphere.Scale bar=500 μm. Pharmacokinetic analyses measuring topotecan levels inthe infused and non-infused hemisphere immediately (t=0) or five hours(t=5) after cessation of drug delivery. For the infused hemisphere,one-way ANOVA, F_((2,20))=38.16, P<0.001, Bonferroni post hoc test,*P<0.01, n=5-9/group. For the non-infused hemisphere, one-way ANOVA,*P<0.05, n=6-9/group. (B) Representative sections and (C) quantificationof optical intensity of UBE3A-YFP in hippocampal regions (CA1, CA2/3,and dentate gyrus=DG) of the topotecan-infused hemisphere or thehemisphere of vehicle-treated mice. *P<0.05, Mann-Whitney Rank Sum Test,n=5/group. (D) Representative sections and (E) quantification ofpaternal UBE3A-YFP in the striatum following i.c.v. infusion oftopotecan. *P<0.05, two-tailed t test, n=4/group. (F) Schematicdepicting schedule for i.t. delivery of topotecan (50 nmol/day for 10 of14 days) and endpoints (arrows) immediately, 4 weeks, and 12 weeks aftercessation of drug treatments. (G,H) Topotecan (i.t.) increased thenumber of UBE3A-YFP-positive spinal neurons compared to vehicle, and theunsilencing of Ube3a-YFP was maintained for at least 12 weeks. One-WayANOVA, F_((5,41))=34.00, P<0.001; Bonferroni post hoc test aftersignificant main effect, *P<0.001, n=7-8/group.

FIGS. 16A-D. Method for detecting paternal UBE3A-YFP protein in neuronsusing high-content screening microscopy. (A) Neurons from paternalUbe3a-YFP mice (m⁺/p^(YFP)) were cultured in 384-well plates and treatedwith small molecules (10 μM) or vehicle (0.2% DMSO) in quadruplicate.Inset depicts drugs applied to a portion of the original 384-well plateused to identify irinotecan as an active. (B) Fluorescence for DAPI(nuclear marker) and UBE3A-YFP in the original plate used to identifyirinotecan as an active. UBE3A-YFP protein signals were amplified usingan anti-GFP antibody. Images show the zoomed inset outlined in (A). Bluebox highlights vehicle control wells and red box highlightsirinotecan-treated wells. (C) 4×4 montaged images of wells were obtainedusing the BD Pathway 855 microscope. This enabled fluorescencedetermination from >1200 individual neurons/well. Using custom writtenalgorithms and Arrayscan software, neuronal nuclei were identified usingthe DAPI channel and detected objects (yellow circles) overlayed ontothe UBE3A-YFP image (red circles). This enabled detection of nuclearUBE3A-YFP fluorescence intensity. Scale bars show 100 μm in montagedimages (left) or 30 μm in zoomed images. (D) Quantitative results(mean±s.e.m.) from quadruplicate wells.

FIG. 17. Autofluorescent compounds. Potential hits, shown in FIG. 13D,were subsequently tested for inherent fluorescence. Wild-type (m⁺/p⁺)neurons were treated for 72 h with 10 μM of compounds followed byimaging to identify possible inherent fluorescence of compounds in the488 excitation/515 longpass emission channel. Fluorescence detectionfrom wild-type neurons demonstrates that the compounds shown in this bargraph exhibited high levels of intrinsic fluorescence in quadruplicatewells of cells, with similar results obtained in two independentexperiments. Bars represent means s.e.m.

FIG. 18. Topoisomerase inhibitors that unsilence UBE3A-YFP do notexhibit inherent fluorescence. All topoisomerase type I and type IIinhibitors that were classified as active in the primary screen weretested for inherent fluorescence in the high-content screen imagingassay. Wild-type (m⁺/p⁺) neurons were treated for 72 h with 10 μM ofcompounds followed by imaging. None of these active compounds increasedfluorescence compared to vehicle-treated neurons. Results represent themean±s.e.m. from four treated wells of neurons. Similar results wereobtained in two independent experiments.

FIGS. 19A-B. Dose response curves and structures for the topoisomerasetype I inhibitor camptothecin and two structurally-related analogs. (A)Dose response curve for camptothecin and lactam E ring camptothecin(inactive). Chemical modification of the camptothecin lactone E ring toa lactam E ring (circles) results in a topoisomerase inactive compound.(B) Dose response curve and chemical structure for 7-ethyl-camptothecin.Results are the mean±s.e.m. from three independent experiments.

FIGS. 20A-B. Dose response curves and structures of the topoisomerasetype I inhibitors 10-hydroxy-camptothecin, 7-ethyl-10-hydroxy-CPT, andirinotecan. (A) Dose response curve and chemical structure for10-hydroxy-camptothecin (CPT). (B) Dose response curve and chemicalstructure for 7-ethyl-10-hydroxy-CPT (SN38) and irinotecan. Structureactivity relationship is apparent between irinotecan and its metaboliteSN38. Results are the mean±s.e.m. from three independent experiments.

FIGS. 21A-B. Dose response curves and structures of the topoisomerasetype I inhibitors rubitecan, belotecan, and silatecan. (A) Dose responsecurve and chemical structure for rubitecan. (B) Dose response curve andchemical structure for belotecan and silatecan. Results are themean±s.e.m. from three independent experiments.

FIGS. 22A-C. Dose response curves and structures of threeindenoisoquinoline (non-camptothecin)-based topoisomerase type Iinhibitors. Dose response curves and chemical structure for (A)NSC706744, (B) NSC725776 and (C) NSC724998. Results are the mean±s.e.m.from three independent experiments.

FIG. 23. Dose response curve and structure of the topoisomerase type IIinhibitor etoposide. Results are the mean±s.e.m. from one experimentperformed in quadruplicate.

FIGS. 24A-B. Dose response curves and structures for the topoisomerasetype II inhibitors ICRF-193 and ICRF-187. Dose response curves andchemical structures for (A) ICRF-193 and (B) ICRF-187 (dexrazoxane).Results are the mean±s.e.m. from two independent experiments, eachperformed in quadruplicate.

FIG. 25. Dose response curve and structure for the topoisomerase type IIinhibitor amsacrine. Results are the mean±s.e.m. from three independentexperiments.

FIGS. 26A-B. The levels of paternal Ube3a unsilencing are proportionalto the treatment time of topotecan. (A) Paternal Ube3a-YFP (m⁺/p^(YFP))neurons were treated with 300 nM topotecan for the times indicatedfollowed by drug removal and replacement with conditioned media; cellswere fixed 72 h after initial drug exposure. (B) UBE3A-YFP fluorescencewas determined from paternal Ube3a-YFP neurons treated with topotecan orvehicle for various durations during a 72 h period. Notice a significantdifference of UBE3A-YFP intensity between 0 and 4, 8, 16, 24, 48 and 72hrs. One Way-ANOVA, F_((7, 48))=147.449, P<0.001; Bonferroni post hoctest after significant main effect indicates comparisons to 0 hr,*P<0.001, n=5-8/time point. Results are the mean±s.e.m, from four wellsof cells, and similar results were obtained in three independentexperiments.

FIG. 27. Bisulfite sequencing of the Ube3a-ATS/Snrpn promoter region incortical neurons following treatment with vehicle or topotecan. Corticalneurons from hybrid CAST/EiJ×C57BL/6 embryos were treated with vehicle(0.1% DMSO) or 300 nM topotecan. Genomic DNA was isolated after 72 h ofdrug treatment and bisulfite conversion was performed. Two primer setswere used to amplify bisulfite treated DNA isolated from corticalcultures: one flanking the first exon of Snrpn and encompassing 13 CpGs,the second to a sequence in the first intron of Snrpn and encompassing14 CpGs (13 for paternal clones, as one CpG is polymorphic). Clones wereidentified as originating from the maternal or paternal chromosome onthe basis of polymorphisms between the CAST/EiJ and C57BL/6 strains.Filled circles represent methylated cytosine residues, open circlesrepresent unmethylated residues. Clones were derived from threeindependent cortical cultures. Efficiency of bisulfite conversion forall samples was greater than 97%.

FIGS. 28A-C. Effects of topotecan on mouse body weight. (A) Normalizedbody weight in mice given vehicle (50 mM tartaric acid in saline) ortopotecan (3.74 or 6.22 μg/h) administered for two weeks byintracerebroventricular (i.c.v.) infusion using mini-osmotic pumps.Kruskal-Wallis one-way ANOVA was tested for vehicle; One-way ANOVA,F_((13,84))=2.532; P<0.01 Bonferroni post hoc test after significantmain effect, *P<0.05 for 3.74 μg/h; One-way ANOVA, F_((13,28))=4.9,P<0.001, Bonferroni post hoc test after significant main effect, *P<0.05for 6.22 μg/h. (B,C) Normalized body weights in mice given vehicle (10%DMSO or 50 mM tartaric acid in 0.9% saline) or topotecan (dose indicatedin figure) by daily intrathecal injection for 10 of 14 days followed byup to four weeks off-drug. For all panels, topotecan or vehicle-treatedgroups were compared to their first day (A) or the average of two daysbefore injection (B,C). For data in panels (B) and (C), Kruskal-Wallisone-way ANOVA with Dunn's post hoc test compared body weights after thefirst day of drug or vehicle injections to the two day average beforeinjections began (represented at Day −1), *P<0.05. The body weight ofmice was measured daily throughout a two (A) or six (B,C) week period.

FIG. 29. Pharmacokinetics in brain. Topotecan levels in the hippocampus(Hip.), striatum (Str.), cerebral cortex (Ctx.), and cerebellum (Cb.) ofthe infused and non-infused hemisphere immediately after cessation ofintracerebroventricular drug delivery (3.74 μg/hr for one week).Topotecan was delivered effectively to the hippocampus, striatum, andcerebral cortex of the infused hemisphere, but not the cerebellum.n=4/group. All data are presented as means±s.e.m.

FIG. 30. Topotecan unsilences the paternal Ube3a-YFP allele in cerebralcortex. Paternal Ube3a-YFP mice were administered vehicle (50 mMtartaric acid) or topotecan (3.74 μg/h) by intracerebroventricular(i.c.v) infusion using mini-osmotic pump for two weeks. In two of fivemice, the cerebral cortex near the infused ventricle exhibited robustunsilencing of paternal Ube3a-YFP following topotecan treatment. Scalebar=100 μm.

FIG. 31. Higher concentrations of topotecan delivered i.c.v. unsilencethe paternal Ube3a-YFP allele in cerebellar Purkinje cells.Ube3a-YFP^(m+/pYFP) mice were administered vehicle (50 mM tartaric acid)or topotecan (21.6 μg/h) by intracerebroventricular (i.c.v) infusionusing mini-osmotic pump for five days. Calbindin-positive Purkinje cellsexpressed paternal Ube3a-YFP following topotecan. Scale bar=100 μm.

FIGS. 32A-C. Topotecan unsilences the paternal Ube3a-YFP allele inneurons. (A) Expression of UBE3A-YFP, the neuronal marker NeuN, and thenuclear marker DRAQ5 following intrathecal injection of vehicle ortopotecan (50 nmol/day for 10 of 14 days) in mice expressing Ube3a-YFPfrom the maternal or paternal chromosome. Scale bar=50 (B) Percentage ofUBE3A-YFP⁺ cells that were NeuN⁺. These data demonstrate that topotecanpredominately unsilences paternal Ube3a-YFP in neurons (93.25%), similarto the predominant expression of maternal Ube3a-YFP in neurons (99.25%).(C) Average pixel intensity of maternal UBE3A-YFP⁺ cells compared topaternal UBE3A-YFP⁺ cells after topotecan treatment. *P<0.001,Mann-Whitney rank sum test.

DETAILED DESCRIPTION OF THE INVENTION

Particular aspects of this invention are explained in greater detailbelow. This description is not intended to be a detailed catalog of allthe different ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure that do not depart from the instant invention.

Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, nucleotidesequences, amino acid sequences and other references mentioned hereinare incorporated by reference in their entirety.

The present invention is based on the development of screening assays toidentify substances (e.g., small molecules) that unsilence imprintedgenes. This invention is further based on the unexpected discovery thattopoisomerase inhibitors can be used to treat genomic imprintingdisorders, such as Angelman syndrome.

Thus, in one embodiment, the present invention provides a method ofinducing expression of Ube3a in a cell, comprising contacting the cellwith an effective amount of a topoisomerase inhibitor, thereby inducingexpression of Ube3a in the cell. In some embodiments, the cell can be acentral neuron, a peripheral neuron, a neuron differentiated from stemcells, a glial cell, an astrocyte, an oligodendrocyte, a microglialcell, and any combination thereof. In some embodiments, the cell can bein a subject (e.g., a human subject) and the Ube3a that is induced isthe paternal allele of Ube3a. Such a subject can be a subject in whichfunction of the maternal allele of Ube3a has been lost and Ube3a proteinproduction is defective in the subject.

In further embodiments of the present invention, a method is provided oftreating a genomic imprinting disorder in a subject, comprisingadministering to the subject an effective amount of a topoisomeraseinhibitor, thereby treating the genomic imprinting disorder in thesubject. In particular embodiments, the genomic imprinting disorder isAngelman syndrome. In some embodiments, the genomic imprinting disorderis Prader Willi syndrome, Beckwith-Wiedemann syndrome, Russell-Silversyndrome, Albright hereditary osteodystrophy, or Turner's syndrome andany combination thereof.

The present invention further provides a method of treating a disorderassociated with an epigenetic modification in a subject, comprisingadministering to the subject an effective amount of a topoisomeraseinhibitor, thereby treating the disorder associated with the epigeneticmodification in the subject. In some embodiments, the disorderassociated with an epigenetic modification can be but is not limited toan autism spectrum disorder, depression, schizophrenia, Rett syndrome,Fragile X syndrome, and any combination thereof.

In some embodiments, the topoisomerase inhibitor of this invention canbe a topoisomerase I inhibitor, which can be, in some embodiments, acamptothecin derivative. A camptothecin derivative of this invention canbe, but is not limited to Belotecan (CKD602), Camptothecin,7-Ethyl-10-Hydroxy-CPT, 10-Hydroxy-CPT, Rubitecan (9-Nitro-CPT),7-Ethyl-CPT, Topotecan, Irinotecan, Silatecan (DB67) and any combinationthereof.

In some embodiments of this invention, the topoisomerase I inhibitor canbe an indenoisoquinoline derivative, which can be but is not limited toNSC706744, NSC725776, NSC724998 and any combination thereof.

In further embodiments of this invention, the topoisomerase inhibitor isa topoisomerase II inhibitor, which in some embodiments can be anacridine derivative, which can be but is not limited to Amsacrine, insome embodiments the topoisomerase II inhibitor can be a podophyllotoxinderivative, which can be but is not limited to etoposide, and in someembodiments the topoisomerase II inhibitor can be a bisdioxopiperazinederivative, which can be but is not limited to ICRF-193, dexrazoxane(ICRF-187) and any combination thereof.

In yet further embodiments of this invention, the topoisomeraseinhibitor can be Resveratrol (PMID: 20304553; PMID: 15796584)⁴¹,Epigallocatechin gallate (PMID: 18293940; PMID: 11594758; PMID:11558576; PMID: 1313232)^(42,43), Genistein (PMID: 17458941)⁴⁴, Daidzein(PMID: 17458941)⁴⁵. Quercetin (PMID: 1313232; PMID: 16950806; PMID:15312049), natural flavones related to quercetin that inhibittopoisomerase, such as acacetin, apigenin, kaempferol and morin (PMID:8567688)⁴⁶⁻⁴⁸, Luteolin (PMID: 12027807; PMID: 16950806; PMID:15312049)⁴⁶; Myricetin (PMID: 20025993)⁴⁹ and any combination thereof.

In certain embodiments of the present invention, the topoisomeraseinhibitor has an efficiency, E_(max), of at least about 1.5, 1.6, 1.7,1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 fold over control.

In certain embodiments, the topoisomerase inhibitor can be aninterfering RNA (RNAi) molecule that targets topoisomerase I,topoisomerase II or both. Nonlimiting examples of RNAi molecules includesmall interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA(miRNA), antisense nucleic acid molecules, and the like as are wellknown in the art. Nonlimiting examples of siRNAs and shRNAs of thisinvention are provided in Table 2. In some embodiments, a zinc fingernuclease, an antibody and/or a ribozyme can be employed to inhibittopoisomerase activity in the methods of this invention.

The present invention additionally provides screening methods toidentify a substance that can unsilence Ube3a expression and/or increaseUbe3a expression. Thus, further provided herein is a screening method toidentify a substance that can increase Ube3a expression, comprisingculturing cells for screening of small molecules, drugs, drug-likecompounds, siRNA constructs, and/or shRNA constructs. In someembodiments, cells are from neurotypical animals, in some instancescells are from mice lacking Ube3a on the maternal allele (Ube3a^(m−/p+))or the paternal allele (Ube3a^(m−/p−)), and in some instances cells arefrom mice expressing Ube3a-YFP knocked into the maternal Ube3a allele,paternal Ube3a allele, or both. These cultured cells are then contactedwith a test substance, which can be but is not limited to a smallmolecule, drug, drug-like compound, siRNA construct, shRNA construct,and/or vehicle control for varying durations as would be known in theart, and then an assessment is made of a test substance-induced increasein Ube3a expression over vehicle control levels through, e.g., direct orantibody-enhanced fluorescence of Ube3a.

Additional approaches to screen for a test substance-induced increase inUbe3a include, e.g., quantitative polymerase chain reaction (PCR) and/orother analogous approaches to identify a test substance-induced increasein Ube3a mRNA expression, and/or immunoblotting or other analogousapproaches as would be well known in the art to identify a testsubstance-induced increase in Ube3a protein expression. Flow cytometrymay be used to identify a test substance that increases Ube3a expressionin cultured cells. In vivo delivery of a test substance can also be usedto determine the ability of a test substance to increase Ube3aexpression; delivery of a test substance can be oral, rectal,transmucosal, topical, intranasal, inhalation (e.g., via an aerosol),buccal (e.g., sublingual), vaginal, intrathecal,intracerebroventricular, intraocular, transdermal, in utero (or in ovo),parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular[including administration to skeletal, diaphragm and/or cardiac muscle],intradermal, intrapleural, intracerebral, and intraarticular), topical(e.g., to both skin and mucosal surfaces, including airway surfaces),and/or transdermal administration, and the like, as well as via directtissue or organ injection (e.g., to liver, skeletal muscle, cardiacmuscle, diaphragm muscle and/or brain).

A test substance identified according to these screening methods thatcan unsilence Ube3a expression and/or increase Ube3a expression can beemployed in the methods of this invention of inducing expression ofUbe3a in a cell and of treating one or more of the various disordersdescribed herein, such as, for example, Angelman syndrome.

Further provides herein is a screening method to identify a substancethat can decrease Ube3a expression, comprising culturing cells forscreening of small molecules, drugs, drug-like compounds, siRNAconstructs, and/or shRNA constructs. In some embodiments, cells can befrom neurotypical animals, in some embodiments, cells can be from micelacking Ube3a on the maternal allele (Ube3a^(m−/p+)) or the paternalallele (Ube3a^(m+/p−)), and in some embodiments, cells can be from miceexpressing Ube3a-YFP knocked into the maternal Ube3a allele, paternalUbe3a allele, or both. These cultured cells are then contacted with atest substance, which can be but is not limited to a small molecule,drug, drug-like compound, siRNA construct, shRNA construct, and/orvehicle control for varying durations as would be known in the art, andthen an assessment is made of a test substance-induced decrease in Ube3aexpression compared to a vehicle control level through, e.g., direct orantibody-enhanced fluorescence of Ube3a.

Additional approaches to screen for a test substance-induced decrease inUbe3a Include quantitative PCR and/or other analogous approaches toidentify a test substance-induced decrease in Ube3a mRNA expression,and/or immunoblotting and/or other analogous approaches to identify atest substance-induced decrease in Ube3a protein expression. Flowcytometry may be used to identify a compound that can decrease Ube3aexpression in cultured cells. In vivo delivery of a test substance canalso be used to determine the ability of a test substance to decreaseUbe3a expression; delivery of a test substance can be oral, rectal,transmucosal, topical, intranasal, inhalation (e.g., via an aerosol),buccal (e.g., sublingual), vaginal, intrathecal,intracerebroventricular, intraocular, transdermal, in utero (or in ovo),parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular[including administration to skeletal, diaphragm and/or cardiac muscle],intradermal, intrapleural, intracerebral, and intraarticular), topical(e.g., to both skin and mucosal surfaces, including airway surfaces),and/or transdermal administration, and the like, as well as via directtissue or organ injection (e.g., to liver, skeletal muscle, cardiacmuscle, diaphragm muscle or brain).

A substance identified according to these screening methods that candecrease Ube3a expression can be used, for example, in a method oftreating disorders associated with increased Ube3a expression, such asautism spectrum disorders. These forms of autism include isodicentricchromosome 15, also known as idic (15), and duplications of chromosome15q11-q13 (dup15q). Potential ‘hits’ identified by this screening methodwould have to go through additional control experiments to verify thatthey are not acting by increasing neuronal death or through generalizedinhibition of protein synthesis. Identified compounds that act todecrease Ube3a levels, without causing cell death or a generalizeddecrease in protein synthesis, can be used as therapeutics for autismspectrum disorders associated with increased Ube3a expression or genedosage.

The present invention also provides various compositions. In someembodiments these compositions can be employed, e.g., in the methodsdescribed herein. Thus, the present invention provides a compositioncomprising, consisting essentially of and/or consisting of atopoisomerase inhibitor and/or other compound of this invention, whichcan be, for example, in a pharmaceutically acceptable carrier.“Pharmaceutically acceptable,” as used herein, means a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject along with the compositions of this invention,without causing substantial deleterious biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. The material would naturallybe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject, as would be well knownto one of skill in the art (see, e.g., Remington's PharmaceuticalScience; latest edition). Exemplary pharmaceutically acceptable carriersfor the compositions of this invention include, but are not limited to,sterile pyrogen-free water and sterile pyrogen-free physiological salinesolution, as well as other carriers suitable for injection into and/ordelivery to a subject of this invention, particularly a human subject,as would be well known in the art.

It is further contemplated that the present invention provides a kitcomprising, consisting essentially of and/or consisting of one or morecompositions of this invention. It would be well understood by one ofordinary skill in the art that the kit of this invention can compriseone or more containers and/or receptacles to hold the reagents (e.g.,topoisomerase inhibitors, etc.) of the kit, along with appropriatebuffers and/or diluents and/or other solutions and directions for usingthe kit, as would be well known in the art. The compositions and kits ofthe present invention can also include other medicinal agents,pharmaceutical agents, carriers and diluents, etc., in any combination.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art.

In the kits of this invention, the compositions can be presented inunit\dose or multi-dose containers, for example, in sealed ampules andvials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of a sterile liquid carrier, for example,saline or water-for-injection prior to use.

Further Definitions

The following terms are used in the description herein and the appendedclaims:

As used herein, “a,” “an,” or “the” can mean one or more than one. Forexample, “a” cell can mean a single cell or a multiplicity of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

As used herein, a “topoisomerase” or “DNA topoisomerase” is an enzymethat plays a role in the replication, repair, genetic recombination andtranscription of DNA. The topoisomerases constitute a group of enzymesthat catalyze the conversion of DNA from one topological form to anotherby introducing transient breaks in one or both strands of a DNA duplex.Topological isomers are molecules that differ only in their state ofsupercoiling. Type I topoisomerase cuts one strand of DNA and relaxesnegatively supercoiled DNA, but does not act on positively supercoiledDNA. Type II topoisomerase cuts both strands of DNA and increases thedegree of negative supercoiling in DNA. These terms include anyanalogues and derivatives of topoisomerases I and II, as are well knownin the art.

A “topoisomerase inhibitor” or “DNA topoisomerase inhibitor” is acompound that interferes with the activity of a topoisomerase. Oneexample of a topoisomerase inhibitor is camptothecin, a natural compoundthat interferes with the activity of topoisomerase I, an enzyme involvedin DNA replication and RNA transcription. Camptothecin and thecamptothecin analogues topotecan and irinotecan are approved forclinical use. Other camptothecin derivatives/analogues include, but arenot limited to, belotecan, rubetecan, and silatecan. Other nonlimitingexamples of a topoisomerase inhibitor include indenoisoquinolinederivatives, acridine derivatives, bisdioxopiperazine derivatives,etoposide, mitoxantrone, lamellarin D, doxorubicin, teniposide,ICRF-193, as well as any other topoisomerase inhibitor now known orlater identified.

As used herein, a “genomic imprinting disorder” means any disordercaused by the mutation or deletion of a gene that is geneticallyimprinted, any disorder caused by alterations of the normal imprintingpattern, and/or any disorder caused by changes in gene dosage of animprinted gene. Nonlimiting examples of a genomic imprinting disorder ofthis invention include Angelman syndrome, Prader Willi syndrome,Beckwith-Wiedemann syndrome, Russell-Silver syndrome, Albrighthereditary osteodystrophy and Turner's syndrome. Any of these disorderscan be treated according to the methods provided herein.

Also as used herein, a “disorder associated with an epigeneticmodification” means a disorder associated with changes of normalchromatin pattern, including but not limited to histone modificationsand DNA methylation. Nonlimiting examples of a disorder associated withan epigenetic modification include autism spectrum disorders, cancer,depression, schizophrenia, Rett syndrome and Fragile X syndrome.

Symptomology of a genomic imprinting disorder and/or a disorderassociated with epigenetic modification can include, but is not limitedto, severe intellectual disabilities, seizures, EEG abnormalities, gaitdisturbances, disrupted sleep patterns, somatosensory deficits, profoundlanguage impairments, abnormal pain sensitivity, and balanceabnormalities, which can be manifested singly and/or in any combinationover time. This it is further contemplated that the present inventionprovides methods of treating one or more of these symptoms in anycombination in a subject, comprising administering to the subject aneffective amount of a topoisomerase inhibitor.

A subject of this invention can be any animal in which genomicimprinting disorders occur and in particular embodiments, is a humansubject, although nonhuman subjects [e.g., animal models of genomicimprinting disorders such as rodents (mice, rats, hamsters, guinea pigs,etc.), pigs, non-human primates] are included within the presentinvention.

A subject of this invention can be “in need of” the methods of thepresent invention, e.g., because the subject has, or is believed at riskfor, a genomic imprinting disorder including those described herein,such as Angelman syndrome and/or is a subject that would benefit fromthe methods of this invention. For example, a subject in need of themethods of this invention can be, but is not limited to, a subjectdiagnosed with, having or suspected to have, or at risk of having ordeveloping a genomic imprinting disorder (e.g., Angelman syndrome).

The term “therapeutically effective amount” or “effective amount,” asused herein, refers to that amount of a topoisomerase inhibitor and/orother compound and/or composition of this invention that imparts amodulating effect, which, for example, can be a beneficial effect, to asubject afflicted with a condition (e.g., a disorder, disease, syndrome,illness, injury, traumatic and/or surgical wound), including improvementin the condition of the subject (e.g., in one or more symptoms), delayor reduction in the progression of the condition, delay of the onset ofthe condition, and/or change in clinical parameters, status orclassification of a disease or illness, etc., as would be well known inthe art.

For example, a therapeutically effective amount or effective amount canrefer to the amount of a topoisomerase inhibitor and/or other compoundand/or composition of this invention that improves a condition in asubject by at least about 5%, e.g., at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or at least about 100%.

“Treat” or “treating” or “treatment” refers to any type of action thatimparts a modulating effect, which, for example, can be a beneficialeffect, to a subject afflicted with a condition (e.g., disorder,disease, syndrome, illness, traumatic or surgical wound, injury, etc.),including improvement in the condition of the subject (e.g., in one ormore symptoms), delay or reduction in the progression of the condition,delay of the onset of the condition, and/or change in clinicalparameters, disease or illness, etc., as would be well known in the art.

By the terms “treat,” “treating” or “treatment of” (or grammaticallyequivalent terms), it is also meant that the severity of the subject'scondition is reduced or at least partially improved or amelioratedand/or that some alleviation, mitigation or decrease in at least oneclinical symptom is achieved and/or there is a delay in the progressionof the condition and/or delay of the onset of a disease or disorder.

By “prevent,” “preventing” or “prevention” is meant to avoid oreliminate the development and/or manifestation of a pathological stateand/or disease condition or disorder or status in a subject.

Exemplary modes of administration of a topoisomerase inhibitor and/orother compound and/or composition of this invention can include oral,rectal, transmucosal, topical, intranasal, inhalation (e.g., via anaerosol), buccal (e.g., sublingual), vaginal, intrathecal,intracerebroventricular, intraocular, transdermal, in utero (or in ovo),parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular[including administration to skeletal, diaphragm and/or cardiac muscle],intradermal, intrapleural, intracerebral, and intraarticular), topical(e.g., to both skin and mucosal surfaces, including airway surfaces, andtransdermal administration, and the like, as well as direct tissue ororgan injection (e.g., to liver, skeletal muscle, cardiac muscle,diaphragm muscle or brain). The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand on the nature of the particular therapeutic compound and/orcomposition that is being used.

For injection, the carrier will typically be a liquid. For other methodsof administration, the carrier may be either solid or liquid. Forinhalation administration, the carrier will be respirable, and willtypically be in solid or liquid particulate form.

Dosages of the topoisomerase inhibitor and/or other compound(s) of thisinvention to be administered to a subject will depend upon the mode ofadministration, the disease or condition to be treated, the individualsubject's condition, the particular therapeutic compound and/orcomposition, and any other agents being administered to the subject andcan be determined in a routine manner according to methods well known inthe art. An exemplary dosage range for a human subject is from about0.001 mg/kg/day to about 500 mg/kg/day. In some embodiments, the dosagerange can be from about 0.01 mg/kg/day to about 100 mg/kg/day and insome embodiments, the dosage range can be from about 0.1 mg/kg/day toabout 10 mg/kg/day.

A nonlimiting example of a method of treating Angelman syndrome in ahuman subject comprises administering to the subject (e.g., in utero,perinatally, postnatally, during infancy, during childhood, duringadolescence, during teen years, during early adulthood, during middleadulthood, during late adulthood and any combination thereof), a dose ofa topoisomerase inhibitor of this invention in the range of about 0.01nmole to about 100 mmole by an intrathecal and/orintracerebroventricular route. In an embodiment in which thetopoisomerase inhibitor is an interfering RNA (RNAi) molecule (e.g.,siRNA; shRNA, etc.), the route of administration could be intrathecaland/or intracerebroventricular and the dose could be in the range ofabout 0.01 mg/kg to about 100 mg/kg and in some embodiments could beabout 0.1 mg/kg to about 10 mg/kg.

In some embodiments, a single administration of a therapeutic compoundand/or composition of this invention may be effective. In otherembodiments, more than one administration (e.g., two, three, four ormore administrations) of the therapeutic compound and/or composition maybe employed to achieve the desired result over a period of variousintervals, e.g., hourly, daily, weekly, monthly, yearly, etc.

The present invention will now be described with reference to thefollowing examples. It should be appreciated that these examples are forthe purposes of illustrating aspects of the present invention, and donot limit the scope of the invention as defined by the claims providedherein.

EXAMPLES Example 1 Small Molecule Screening

Initial studies identified DNA topoisomerase inhibitors as the firstclass of small molecules that can unsilence an imprinted gene. This isboth unprecedented and significant. Topoisomerase inhibitors, used toselectively kill rapidly dividing tumor cells, have never before beenshown to unsilence imprinted genes. These findings are thus doublyimportant, as they identify a novel potential therapeutic use for anexisting drug class, as well as a potential strategy that could beapplied to treat Angelman syndrome, as well as other disorders involvingimprinted genes (e.g., Prader-Willi syndrome, Beckwith-Wiedemannsyndrome, Russell-Silver syndrome, and others) (Morison and Reeve, 1998;Butler, 2009). In some embodiments, the methods of the present inventionallow for the identification of substances (e.g., small molecules) thatoptimally unsilence Ube3a in vitro and in vivo. Such a small moleculeapproach can target neurons throughout the central nervous system (CNS)(FIGS. 8-10).

Small molecules that induce expression of paternal Ube3a translate intoan effective treatment for Angelman syndrome (FIGS. 1-2), because thesilenced paternal Ube3a allele remains structurally intact in neuronsfrom individuals with the syndrome. In embodiments of this invention, ahigh-content and high-throughput assay (FIG. 3) has been developed, bywhich numerous compounds, including irinotecan and topotecan, have beenidentified that can unsilence the paternal Ube3a allele (FIGS. 4-10).All validated hits to date are DNA topoisomerase inhibitors.

The high-content screening method was developed by taking advantage oftransgenic Ube3a-YFP knock-in mice and high content imaging microscopy(Dindot et al., 2008). In the transgenic mice, Ube3a is fused to yellowfluorescent protein (YFP), thus allowing the visualization of Ube3aprotein expression using fluorescence detection. Using neurons culturedfrom these mice, a cell-based screening assay was developed that canidentify small molecules that unsilence and upregulate expression ofpaternal Ube3a protein in neurons (FIGS. 2-3). Importantly, this smallmolecule screening assay preserves paternal silencing of Ube3a anddistinguishes between silenced and active Ube3a alleles (FIG. 3). Thisassay has been optimized for the 384-well plate format, enabling thequantification of Ube3a-YFP protein levels in primary neurons usingcustomized algorithms and a BD-Pathway 855 high-content imagingmicroscope platform.

This assay has outstanding characteristics (e.g., DMSO stability,Z′-score, reproducibility) for small-molecule screening. Specifically,to validate that this assay was suitable for screening, a Z′-score wascalculated based on maternal positive and paternal negative Ube3asignals (FIG. 3) and their standard deviation. The highest Z′-score isone, and >0.5 is considered excellent. Thus, the calculated Z′ score of0.56 for this high-content fluorescence microscopy screen indicates thatthis approach has excellent metrics for a full-scale high-content-basedscreen (Zhang et al., 1999). To assess DMSO stability, it wasdemonstrated that 72 h treatments of vehicle containing up to 1% DMSOhad no effect on number, survival, or expression of neurons containingmaternal Ube3a-YFP. Therefore, the 0.2% DMSO in media planned in thesescreening studies should have no adverse effect on neuronal health orsurvival.

Neurons are cultured from mice expressing Ube3a-YFP on the paternal, butnot maternal allele. Drug-induced changes in YFP fluorescence serve as agauge for unsilencing of the paternal Ube3a allele. Using this smallmolecule screening approach, it was discovered that the topoisomeraseinhibitor irinotecan unsilences and upregulates paternal Ube3a protein(FIG. 4). It was subsequently determined in dose-response studies thatirinotecan and the closely related topoisomerase inhibitor topotecan,both unsilence and upregulate paternal Ube3a protein (FIG. 7).Additional studies using the screening platform confirmed compoundactivities and have identified several distinct topoisomerase inhibitorcompounds that unsilence and upregulate paternal Ube3a protein incortical neurons (Table 1).

Methodology Details.

In all screening assays, primary cortical neurons from Ube3a-YFP miceare isolated from embryonic day 15.5 animals, and the genotype ofanimals is determined to identify mice that encode the Ube3a-YFPtransgene on the paternal allele. Freshly isolated cortical neurons areseeded into 384 well plates and cultured for 7 days. At day in vitroseven, screening is performed using multiple chemical libraries andcompound concentration of 10 μM in 0.2% DMSO vehicle. After 72 h of drugexposure (run in quadruplicate wells), neurons are fixed with 4% paraformaldehyde and processed for immunofluorescence using ananti-GFP-Alexa 488 conjugated antibody to enhance the signal ofUbe3a-YFP in neurons. For image analyses, individual wells ofimmunofluorescence-processed plates are imaged fro DAPI and GFPfluorescence using the BD-Pathway 855 high-content imaging microscope.Ube3a-YFP fluorescence intensities in individual neurons are determinedin drug-treated wells and normalized to 0.2% DMSO-treated (i.e., vehiclecontrol) wells using custom written macros and algorithms using NIHImage J and Arrayscan Cell Profiler software programs. These imageanalyses enable masking of nuclei in individual neurons anddetermination of Ube3a-YFP fluorescence intensity in the nuclei ofindividual neurons. Drug-induced increases of >50% are initially binnedas screening ‘hits’ if the increases are consistently observed acrossmultiple runs and if no apparent toxicities are observed by assessingnuclear structure of neurons co-stained with DAPI. Effective ‘hit’compounds are validated in formal dose-response experiments to determinerelative compound potencies and efficacies.

For these studies, DIV 7 primary neurons encoding paternal Ube3a-YFP aredose treated with hit compounds across seven orders of magnitude in fulland half log molar concentrations (3 pM to 30 μM). Ube3a-YFPfluorescence intensities in living neurons are determined again byhigh-content imaging microscopy after 72 h treatments. The dose responseresults are analyzed by least squares sigmoidal dose-response curvefitting models using Graphpad Prism 5.0 (Graphpad Software, Inc.). Thecalculated EC₅₀ values (potencies) and Y value top plateau (estimatedefficacies or E_(max)) enable comparative analyses of the relativepotency and efficacy of the identified compounds. To control forpotential false positive ‘hit’ compounds that might have inherentcompound fluorescence, cortical neurons from wildtype mice (cells thatdo not encode Ube3a-YFP) are also treated to determine if ‘hit’compounds exhibit inherent fluorescence. None of the identifiedcompounds described display any inherent fluorescence in wildtypeneurons.

‘Hits’ are subsequently evaluated in formal dose-response studies todetermine relative compound potencies and efficacies, using thehigh-content imaging approaches described herein. These experimentsenable the identification of the most potent and efficacious smallmolecule compounds for subsequent testing in vivo. The calculated EC₅₀values (potencies) and relative efficacies guide structure-activitystudies and formal rank-order-potency evaluations. The most efficaciousand potent compounds are evaluated in time-course experiments in whichdrug treatments of neurons are extended from 7 to 21 DIV, with drugreplacement every 72 hours. These studies determine the chronic effectsof the compounds in vitro, providing key information regarding thetemporal peak of paternal Ube3a-YFP expression during drug treatmentsand whether expression is maintained over time in culture.

The most efficacious small molecules are tested in dose-response studies[e.g., 0, 2, 10, 20, and 200 nmol in 5 μl for intracerebroventricular(i.c.v.) injections] in mice expressing paternal Ube3a-YFP (see FIG. 9).The ability of these compounds to upregulate paternal Ube3a in maternalUbe3a-null mice (Ube3a^(m−/p+)) is also evaluated. Neurons in theneocortex, hippocampus, spinal cord, and dorsal root ganglia areexamined for recovery of Ube3a-YFP expression because it has beendemonstrated that Ube3a is imprinted in these regions (FIGS. 8 and 9).

Example 2 Quantitative RT-PCR and Western Blot Analysis to Confirm thatCompounds Unsilence the Paternal Ube3a-YFP Allele

The fluorescence imaging assay of Ube3a-YFP described above is an idealscreening platform to identify active small molecules that unsilenceUbe3a; however, it is important to use an orthogonal assay to verifythat these topoisomerase inhibitors increase endogenous Ube3a mRNA andprotein in neurons. To this end, RT-PCR analyses determined thattreatment of neurons with irinotecan significantly increased the levelof paternal Ube3a-YFP mRNA, indicating irinotecan increases Ube3a-YFPmRNA expression by unsilencing the paternal allele (FIG. 5A). Similarly,immunoblot analyses determined that irinotecan treatment of neuronalcultures significantly increases paternal Ube3a-YFP protein levels (FIG.5B). Finally, to confirm that endogenous Ube3a is upregulated by thetopoisomerase inhibitor, immunoblot analyses also determined thatirinotecan treatment of cortical neurons from Angelman syndrome modelmice upregulated paternal Ube3a protein levels (FIG. 6). These findingsconfirm, using orthogonal assays of RT-PCR and immunoblotting, that thetopoisomerase inhibitor irinotecan upregulates paternal Ube3a-YFP incortical neurons. Similar verification experiments will be performedwith other identified hit molecules from screening.

Real Time PCR.

Primary cortical neurons from Ube3a-YFP mice were isolated at embryonicday 13.5 to 15.5 and cultured in 6 well poly-D-lysine-coated plates, andthe genotype of animals was determined to identify animals that encodethe Ube3a-YFP transgene on the paternal allele. At day in vitro seven,primary cortical neurons that encode paternal Ube3a-YFP were treatedwith 14 μM irinotecan or 0.2% DMSO for 72 h. Total mRNA was isolated byguanidinium thiocyanate-phenol-chloroform extraction using the Trizolreagent (Invitrogen Inc.). Total mRNAs from samples were converted intocDNA and subjected to real-time PCR (RT-PCR) analyses to quantifyUbe3a-YFP transcript levels using oliognucleotide primers against Ube3a(Forward sequence: caaaaggtgcatctaacaactca; reverse sequence:ggggaataatcctcactctctc) and total transcript reads were normalized toOaz1 (Forward sequence: cctgagggcagtaaggacag; reverse sequence:ccaagaaagctgaaggttcg).

Western Blotting.

Embryonic day 13.5 to 15.5 mice that encode the paternal Ube3a-YFPtransgene or Ube3a^(m−/p+) mice (i.e., AS model mice) were identifiedand primary cortical neuronal cultures were obtained and plated in 6well plates. At day in vitro seven, primary cortical neurons weretreated with 10 μM irinotecan or 0.2% DMSO for 72 h and total proteinlysates were obtained by lysis buffer (1% Triton X-100, 5 mM EDTA, 0.15MNaCl, 10 mM Tris-HCl, pH 7.5, phosphatase inhibitor cocktails 1,protease inhibitor cocktail). To assess Ube3a-YFP protein levels, 7.5 ugof total protein lysates from Ube3a-YFP neurons were separated by 8%SDS-polyacrylamide gel electrophoresis, proteins were transferred tonitrocellulose membranes and immunoblotting was performed using a rabbitanti-GFP antibody (Novus) and Alexa Fluor 680 goat anti-rabbit IgG(Invitrogen). Ube3a-YFP protein bands were visualized by a LiCOR systemand to control for protein loading, Ube3a-YFP protein levels werenormalized to actin level detected in each sample. To assess Ube3aprotein level, immunoblotting was performed using rabbit anti-Ube3aantibody (Abeam) and Alexa Fluor 680 goat anti-rabbit IgG (Invitrogen).

Example 3 In Vivo Studies

To determine if topoisomerase inhibitor compounds are also active invivo, preclinical testing of compounds was performed using miceexpressing paternal Ube3a-YFP. For these studies, Ube3a-YFP micereceived topotecan or irinotecan by intracerebroventricular (ICV) orintrathecal (IT) delivery, respectively, and detection of paternalUbe3a-YFP in spinal cord or brain was done using immunofluorescencedetection. These in vivo studies determined that IT treatments of micewith irinotecan increased paternal Ube3a-YFP protein in the spinal cordof mice (FIG. 8) and ICV infusions of topotecan increased paternalUbe3a-YFP protein levels throughout the brain (FIG. 9). Fordose-response studies, mice receive increasing concentrations ofirinotecan or topotecan.

Methodology Details.

Immunohistochemistry, quantitative PCR, and Western blotting methods areused to determine if candidate hit molecules upregulate paternalUbe3a-YFP after in vivo treatments. Comparisons of Ube3a levels betweendrug- and vehicle-treated paternal Ube3a-YFP mice are assessed. A totalof six mice for each treatment at 3-6 months of age are used to identifyoptimal dosage for potent and effective candidate drugs. This age rangewas chosen based on successful pilot experiments (FIGS. 8 and 9). Drugsare initially administered for 4 days, based on the success of thisdosing regimen in pilot experiments (FIG. 8), although testing lowerdoses for longer periods and higher doses for shorter periods of time isalso informative.

Compounds are administered using three approaches:intracerebroventricularly (i.c.v.), intraperitoneally (i.p.), andintrathecally (i.t.). Each method has its own advantages: i.p.injections are non-invasive and can assess blood-brain barrier (BBB)penetration; i.c.v. injections bypass the BBB to allow direct assessmentof drug efficacy in brain; and i.t. injections provide the easiest andmost efficient approach for simultaneously assessing drug permeabilityin the spinal cord, where there is a BBB, and in dorsal root ganglionneurons, where there is no BBB. In addition, all delivery methods couldbe used to treat symptoms associated with Angelman syndrome. Drugs areinitially administered intracerebroventricularly (i.c.v.) andintraperitoneally (i.p.). For compounds like topotecan and otherapproved medications for which maximum tolerated dose regimens are knownin mice (Kim et al., 1992), standard doses used in murine models areused (e.g., 2 mg/kg for topotecan; 20-50 mg/kg irinotecan; Kunimoto etal., 1987). Some of these initial compounds are FDA-approved, and thuswell characterized, but for less well characterized compounds low dosesare used initially, CNS levels are determined, and subsequent desirablelevels are estimated based on EC₅₀ for activity in neurons and guidancefrom veterinary staff on drug tolerance. Drugs are also administeredintrathecally (Lt.), starting at low concentrations (e.g., 2 nmol) andincreasing concentrations in small increments so that drug tolerance canbe assessed.

For proof-of-principle of in vivo effect of irinotecan, intrathecalinjection was performed. 5 ul of Irinotecan was injected intounanesthetized mice using the direct lumbar puncture method. A softcloth is placed over the head and upper body of the animal to keep himcalm. The experimenter grasps the pelvic girdle of mouse with his thumband forefinger. Next, the experimenter traces the spinal column of themouse with a needle. The disposable 30 gauge ½ inch needle is connectedto a 50 ul Luer-hub Hamilton syringe. As long as the experimenter sensesthe bones of the spinal column, the angle of syringe is lowered at 45degree to the horizontal. The experimenter inserts the needle 1 mmfurther to the spinal column. Following the 5 ul injection, the syringeis rotated slightly and removed. Irinotecan is dissolved in 0.9% salinewith 10% DMSO. Three different dosages of Irinotecan are used. They are100, 500 and 1000 nmole. Paternal Ube3a-YFP (+/Ey) mice are usedthroughout these experiments.

It was found that the paternal Ube3a-YFP allele is silenced in spinalneurons (FIG. 8A). Paternal Ube3a-YFP is also silenced in proprioceptiveand mechanoreceptive sensory neurons (located in the dorsal rootganglia; DRG). Drug-like small molecules can readily be delivered tospinal and DRG neurons via intrathecal injections. Strikingly,intrathecal injection of either irinotecan (FIG. 8B; 5 μl injections of1000 nmol/day for 5 days) or topotecan (FIG. 8C; 5 μl injections of 200nmol/day for 6 days) into mice in vivo unsilenced the paternal Ube3a-YFPallele in spinal neurons and in DRG neurons. Importantly, neuronsappeared normal in drug-treated mice as evidenced by counterstainingwith a pan-neuronal marker (FIGS. 8D-F).

In order to unsilence paternal Ube3a in vivo with an emphasis on brain,I.C.V. with osmotic minipump was performed. I.C.V. alone method wasperformed first to identify the coordinate of injection and the potencyof topotecan. Paternal Ube3a-YFP mice were anesthetized withKetamine/Xylazine (120 mg/kg; 9 mg/kg) and positioned in a stereotaxichead frame. The scalp is shaved and cut, and the skull exposed. Thelocal anesthetic (Bupivacaine, 2.5 mg/ml) is applied on the top of skulland mineral oil is applied on the eyes of the mice. Acetone is appliedon the skull to remove any lipid tissues on the skull as well as to drythe skull surface for optimal adhesion. Next, a cannula (Brain InfusionKit 1, DURECT Corporation) is positioned into a lateral ventricle at thefollowing coordinates (−0.3 mm A/P, +/−1.0 mm M/L, −3.0 mm D/V). Thefree end of the cannula is connected to an Alzet osmotic minipump via a2.5-cm-long piece of polyethylene (PE) tubing (cat. no. 0007750, DURECTCorporation). The osmotic minipump and the connecting PE tubing arefilled with 16.34 mM topotecan (CPT06, Molcan) dissolved in 50 mMtartaric acid with saline (or control solution without drug). The filledpumps are incubated in sterile saline at 37° C. for at least 4 to 6hours for priming before being implanted under the dorsal skin of themouse's back. A needle driver is used to create a subcutaneous pocket inthe scapula region to accommodate filled pumps. The cannula base and theattached piece of PE tubing are fixed to the skull with loctitecyanoacrylic. The incision site is closed with prolene suture. Duringand after surgery, mice are placed on a heating pad. Body weight of miceis recorded daily to assess mouse health. Seven days following minipumpimplantation, mice are sacrificed following pentobarbital overdose (150mg/kg, I.P.) and brains are removed for immunofluorescence staining orWestern blot analysis.

It was found that irinotecan delivered intracerebroventricularly(i.c.v.) significantly increased Ube3a levels in Angelman syndrome modelmice, and that topotecacan delivered i.c.v. dramatically unsilencespaternal Ube3a-YFP (FIG. 9). These results indicate that irinotecan andtopotecan unsilence Ube3a in vivo.

These in vivo studies indicate that topoisomerase inhibitors areeffective at unsilencing the paternal Ube3a allele in individuals withAngelman syndrome. In the present invention, 14 DNA topoisomeraseinhibitors are identified that effectively unsilence paternal Ube3a (2are FDA approved, 12 are topoisomerase I inhibitors, and 2 aretopoisomerase II inhibitors).

Angelman syndrome model mice have a number of behavioral phenotypesconsistent with Angelman syndrome in humans, including epilepsy,learning defects, and motor abnormalities (Jiang et al., 1998; Miura etal., 2002; Weeber et al., 2003; van Woerden et al., 2007). Since Ube3ais imprinted in proprioceptive neurons, and proprioceptive neuronscoordinate movement, these studies suggest the possibility that themotor and coordination symptoms associated with Angelman syndrome mightbe due to altered proprioceptor function, instead of altered cerebellarfunction as is commonly thought. In addition, these mice exhibitdeficits in synaptic plasticity (long-term depression, LTD, andlong-term potentiation, LTP) and ocular dominance plasticity (Jiang etal., 1998; Yashiro et al., 2009; Sato and Stryker, 2010). Thesewell-characterized electrophysiological and behavioral phenotypes inAngelman syndrome model mice can be used to test for drug-inducedrecovery of function.

Example 4 Persistence of Drug-Induced Upregulation of Paternal Ube3a

To assess if a single topoisomerase inhibitor treatment regimen resultsin long-lasting effects on paternal Ube3a, Ube3a-YFP mice wereadministered two weeks of daily intrathecal injections of topotecan (100nmol/day on 10 of 14 days), and spinal cords were isolated and sectionedand then processed for immunofluorescence detection of Ube3a-YFP. In aseparate experiment, mice were similarly administered 100 nmol/day overa two week period, followed by drug washout for 14 days, and spinalcords were isolated and processed for immunofluorescence detection ofUbe3a-YFP. These experiments determined that paternal Ube3a-YFP isunsilenced in a subset of neurons and that 14 days after cessation oftopotecan injections, unsilencing of paternal Ube3a persists, suggestingthat the loss of Ube3a imprinting is long lasting subsequent totopoisomerase treatments (FIG. 10) and may be permanent.

Studies will be done to determine whether a single dosing regimen canunsilence Ube3a permanently. Using optimized delivery and dosing,Ube3a-YFP levels will be assessed immediately, 2 weeks, 2 months, and 1year after drug treatment (6-8 mice/drug; comparisons will be made tovehicle-treated mice).

Example 5 Age-Dependence of Ube3a Upregulation

Studies will be done to determine whether restoring Ube3a levels inadulthood will be sufficient to fully recover behavioral deficits inAngelman syndrome model mice, or whether Ube3a levels must be restoredearlier in life. Thus, assessments will be made of drug-inducedincreases in Ube3a-YFP, compared to vehicle controls, in mice treated(1) in utero (by intrauterine injection at non-toxic concentrations ofdrugs at E15.5, an age after the maternal imprint has been established);(2) at postnatal day 10 (P10, an age before electrophysiological defectsin the brain are observed; Yashiro et al., 2009); (3) at P20 (an agewhen synaptic deficits are beginning to be observed); and (4) in adult(>P60) mice.

Example 6 Upregulation of Paternal Ube3a in Angelman Syndrome Mice

Ube3a maternal-null mice are the best model of Angelman syndrome.Studies will be done to define an optimized drug regimen capable ofincreasing paternally-coded Ube3a protein in Ube3a^(m−/p+) mice.

Example 7 Comparison of Ube3a m/p+ Mice Given Drug or Vehicle TreatmentsIn Vivo Beginning on P20 or P60

Comparisons will be made of Ube3a^(m−/p+) mice given drug or vehicletreatments in vivo beginning on P20 or P60, using the dosing regimenoptimized as described herein. Additional comparisons will be made towildtype littermates (Ube3a^(m+/p+) mice) receiving vehicle treatments.Recovery of function will be assessed as described herein uponcompletion of drug treatment.

Example 8 RNA-Interference (RNAi) Approaches

Topoisomerase inhibitors are shown herein to unsilence paternal Ube3a,suggesting topoisomerase enzymes regulate Ube3a imprinting. Transientknock-down of topoisomerase I protein is a complementary method to testif loss of topoisomerase function unsilences Ube3a. To determine if lossof topoisomerase function by protein knockdown also results inunsilencing of Ube3a, RNA-interference (RNAi) for topoisomerase I wasassessed. Cultured cortical neurons from the Ube3a-YFP transgenic micewere treated with increasing concentrations of lentivirus-based shRNAtargeting topoisomerase I (Topo I). After five days of treatment withsiRNA, total protein lysates were obtained and Ube3a-YFP protein levelswere determined by Western blotting. Delivery of lentiviruses thatcontain shRNA for topoisomerase I increased Ube3a protein levels incortical neurons indicating topoisomerase I knockdown increases Ube3aexpression (FIG. 12).

In further studies, cultured cortical neurons from the mice describedherein will be treated with lentivirus-based shRNA targetingTopoisomerase I (Topo I), Topo IIa, Topo IIb, non-targeting shRNA(scrambled negative control), vector only (control), or GFPshRNA(control). In some experiments, to complement shRNA approaches, siRNAfor RNAi-mediated knockdown will be alternatively used. For theseexperiments, cultured cortical neurons will be transfected with siRNAtargeting Topo I, Topo IIa, or Topo IIb using lipofectamine 2000.Scrambled siRNA will be used as a negative control. For all RNAiexperiments, high-content imaging and Western blots will be used toassess Ube3a expression following topoisomerase knockdown. Similarapproaches can be used to validate that topoisomerase expression andfunction is required for Ube3a imprinting and silencing.

Example 9 Methylation Assays

Because the allele-specific activation/silencing of Ube3a is thought tooccur through differential DNA methylation (Glenn et al., 1993;Sutcliffe et al., 1994; Glenn et al., 1997), studies will be carried outto determine whether topoisomerase inhibitors alter DNA methylation. Todo this, DNA methylation status within the Angelmansyndrome/Prader-Willi imprinting centers will be determined followingdrug (or vehicle) treatment of cortical neurons using the sodiumbisulfite conversion method (Huang and Akbarian, 2007). In addition, theSequenom MassARRAY platform can be used to identify and quantifydrug-induced changes across differentially methylated regions as well aspyrosequencing approaches to further define regions of differentialmethylation.

Example 10 Histone Modification Assays

Because histone modifications provide another mechanism to alterimprinting status (Soejima and Wagstaff, 2005; Kim and Huibregtse,2009), studies will be carried out to examine whether histonemodifications occur with active small molecules (compared to vehicletreatments). Levels of H3K4 m3 and H3K27me3 within the relevantimprinting center will be quantified by SYBR-based RT-PCR withcustom-made primers.

Example 11 Deep Sequencing

Studies will be conducted to test whether the compounds of thisinvention alter neuronal gene expression. Total brain RNA will beextracted from drug- or vehicle-treated neurons taken from reciprocalprogeny of the CAST/EiJ and BALB/cByJ mouse strains. These divergentmouse strains are amenable to single nucleotide polymorphism (SNP)analyses, allowing for assessment of transcriptional changes in bothimprinted and non-imprinted genes, cDNA will be examined using theIllumina Genome Analyzer (Wang et al., 2008).

Example 12 Synaptic Plasticity Ex Vivo

Ube3a^(m−/p+) mice lack normal synaptic plasticity (LTD/LTP) in visualcortex (Yashiro et al., 2009), and studies will be carried out todetermine whether drug treatments can restore normal plasticity. Fieldpotential recordings will be used to assess synaptic plasticity invisual cortex as described (Yashiro et al., 2009). Field EPSPs (fEPSPs)will be generated in visual cortex slices by providing extracellularlayer 4 stimulation and recording in layers 2/3 (Kirkwood et al., 1993;Philpot et al., 2007). Changes in synaptic strength after 1, 20, 40, or100 Hz stimulation will be assessed by comparing changes in the fEPSPamplitude.

Example 13 Synaptic Plasticity In Vivo

Ocular dominance plasticity is absent in Ube3a^(m−/p+) mice (Yashiro etal., 2009). To examine in vivo cortical plasticity in drug- andvehicle-treated mice, chronic visually-evoked potential (VEP) recordingswill be obtained in unanesthetized mice to assess ocular dominanceplasticity as described (Yashiro et al., 2009).

Example 14 Behavioral Recovery

Angelman syndrome mice have quantifiable deficits in behavior, includingpoor motor performance, a propensity for seizures, abnormal gait, anddeficits in context-dependent fear learning and spatial learning (Jianget al., 1998; van Woerden et al., 2007). To assess the likelytherapeutic value of candidate molecules, evaluations will be done todetermine the extent to which candidate molecules ameliorate behavioraldeficits in Ube3a^(m−/p+) mice. A total of 10-12 mice for each treatment(drug- or vehicle-treated) and genotype (Ube3a^(m−/p+) or Ube3a^(m+/p+))will be assessed by behavioral tests, in a blinded manner. Bar-crossing,footprint analysis, and rotating rod tests will be used to assess motorfunction of mice. Seizure susceptibility in response to cage gratingwill also be tested. Gait will be measured by assessing stride lengthand forepaw/hind paw overlap as described (Trushina et al., 2006).Spatial learning will be assessed using the Morris water maze (FIG. 11),and data will be recorded, including the time taken to reach platform,the time spent in each quadrant of the pool, and total distancetraveled. Context-dependent fear learning will be determined usingpreviously described approaches (Jiang et al., 1998).

Example 15 Additional Data/Observations

Preliminary Affymetrix GeneChip data indicate that there are notlarge-scale expression changes when cultured neurons are treated withirinotecan (e.g., <1% of the genes on chip showed a 1.5× fold increase;and <2% showed a decrease). Both irinotecan and topotecan areFDA-approved chemotherapeutics (Koster et al., 2007). These drugs areused clinically and are in trials for treating brain tumors (Feun andSavaraj, 2008). This makes it unlikely that any epigenetic changescaused by topoisomerase inhibitors would have insurmountable toxicities.

In support of the idea that effective concentrations of irinotecan donot increase cell death, (1) a similar number of pyknotic nuclei weredetected in vehicle- and irinotecan-treated cultures, and (2) noevidence for drug-induced increases in apoptotic pathways was observedby microarray. To further verify that there are no significantdrug-induced increases in cell death (compared to vehicle controls),immuncytochemical assays of apoptosis (e.g. TUNEL assays) will beperformed. Importantly, topoisomerase inhibitors have tolerable sideeffects when administered chronically to treat cancer in adults andchildren (Schellens et al., 1996; Stewart et al., 1996; Creemers et al.,1997; Vassal et al., 1998; Rose et al., 2006). With appropriate dosing,it might be possible to unsilence Ube3a while minimizing these sideeffects. The tolerance of these FDA-approved drugs in humans is wellestablished (Seiter, 2005; Hartmann and Lipp, 2006). The preliminarydata shown herein suggest that the drug-induced loss of paternal Ube3aimprinting may be permanent (FIG. 10). If the loss of imprinting isindeed permanent, then a brief drug treatment regimen may be all that isneeded for a lifelong therapeutic effect.

Studies described herein demonstrate that irinotecan can cross the bloodbrain barrier (BBB) (FIGS. 8 and 10). Moreover, consistent with findingsthat topotecan has greater potency (FIG. 7), stability, and permeabilityacross the BBB than irinotecan (Herben et al., 1996; Herben et al.,1998; Motl et al., 2006), studies described herein show that topotecanis even more effective at unsilencing paternal Ube3a in spinal cord(compare FIGS. 8B and 8C). It was also determined that infusions oftopotecan directly into the brain, thereby bypassing the BBB,upregulates paternal Ube3a throughout the brain (FIG. 9).

It is expected that cellular plasticity and behavioral deficits inUbe3a^(m−/p+) mice can be rescued. A failure to recover behavioraldeficits may be overcome by dosage adjustment, increasing the time ofdrug treatment, or by combining different candidate molecules. It isalso expected that drug-induced therapies might provide some degree ofbenefit regardless of the age treatment is begun, as has been observedin other neurodevelopmental disorders (Ehninger et al., 2008). Insupport of the idea that mental retardations can be overcome later inlife, the late-onset reinstatement of MeCP2 can rescue deficitsassociated with Rett syndrome, a mental retardation and epigeneticdisorder (Guy et al., 2007).

Inappropriately high gene dosage seems unlikely, however, for thefollowing reasons. (1) It is difficult to increase Ube3a levels muchabove normal levels, likely because Ube3a is an E3 ubiquitin ligase thattargets itself for proteasomal degradation (Crinelli et al., 2008). Thisplaces an upper limit on Ube3a protein levels. (2) At least two of theeffective topoisomerase inhibitors are FDA-approved and widely usedclinically. Thus, regardless of changes in gene dosage, the tolerance ofthese compounds in humans is already well established. (3) Neurons arethe only class of cells that exhibit paternal silencing of Ube3a; thusany small molecules that unsilence the paternal allele are likely tohave their largest effects in neurons. (4) Because the maternal Ube3aallele is deleted or mutated in individuals with Angelman syndrome,paternal unsilencing of Ube3a is only capable of producing mono-allelicactivation of the gene. (5) Dosing of the compounds of this inventioncan be regulated to minimize inappropriately high expression levels ofUbe3a in neurons.

REFERENCES FOR EXAMPLES 1-15

-   Abbas A I, Urban D J, Jensen N H, Farrell M S, Kroeze W K,    Mieczkowski P, Wang Z, Roth B L (2010) Assessing serotonin receptor    mRNA editing frequency by a novel ultra high-throughput sequencing    method. Nucleic Acids Res 38:e118.-   Akbarian S, Huang H S (2009) Epigenetic regulation in human    brain-focus on histone lysine methylation. Biol Psychiatry    65:198-203.-   Albrecht U, Sutcliffe J S, Cattanach B M, Beechey C V, Armstrong D,    Eichele G, Beaudet A L (1997) Imprinted expression of the murine    Angelman syndrome gene, Ube3a, in hippocampal and Purkinje neurons.    Nat Genet 17:75-78.-   Alexander G M, Rogan S C, Abbas A I, Armbruster B N, Pei Y, Allen J    A, Normeman R J, Hartmann J, Moy S S, Nicolelis M A, McNamara J O,    Roth B L (2009) Remote control of neuronal activity in transgenic    mice expressing evolved G protein-coupled receptors. Neuron    63:27-39.-   Arn P H, Williams C A, Zori R T, Driscoll D J, Rosenblatt D S (1998)    Methylenetetrahydrofolate reductase deficiency in a patient with    phenotypic findings of Angelman syndrome. Am J Med Genet 77:198-200.-   Bacino C A, Peters S, Goddard-Finegold J, Smiith O, O'Brien W,    Madduri N, Glaze D, Shinawi L, Beaudet A (2003) A randomized    therapeutic trial in Angelman syndrome using betaine and folic acid.    Am J Hum Genet 73 (suppl.):334.-   Bomgaars L, Berg S L, Blaney S M (2001) The development of    camptothecin analogs in childhood cancers. Oncologist 6:506-516.-   Browne C E, Dennis N R, Maher E, Long F L, Nicholson J C,    Sillibourne J, Barber J C (1997) Inherited interstitial duplications    of proximal 15q: genotype-phenotype correlations. Am J Hum Genet    61:1342-1352.-   Butler M G (2009) Genomic imprinting disorders in humans: a    mini-review. J Assist Reprod Genet. 26:477-486.-   Carol H, Houghton P J, Morton C L, Kolb E A, Gorlick R, Reynolds C    P, Kang M H, Maris J M, Keir S T, Watkins A, Smith M A, Lock R    B (2010) Initial testing of topotecan by the pediatric preclinical    testing program. Pediatr Blood Cancer 54:707-715.-   Clayton-Smith J, Laan L (2003) Angelman syndrome: a review of the    clinical and genetic aspects. J Med Genet 40:87-95.-   Collins I, Weber A, Levens D (2001) Transcriptional consequences of    topoisomerase inhibition. Mol Cell Biol 21:8437-8451.-   Cook E H, Jr., Lindgren V, Leventhal B L, Courchesne R, Lincoln A,    Shulman C, Lord C, Courchesne E (1997) Autism or atypical autism in    maternally but not paternally derived proximal 15q duplication. Am J    Hum Genet 60:928-934.-   Corbett K D, Berger J M (2004) Structure, molecular mechanisms, and    evolutionary relationships in DNA topoisomerases. Annu Rev Biophys    Biomol Struct 33:95-118.-   Creemers G J, Gerrits C J, Eckardt J R, Schellens J H, Burris H A,    Planting A S, Rodriguez G I, Loos W J, Hudson I, Broom C, Verweij J,    Von Hoff D D (1997) Phase I and pharmacologic study of oral    topotecan administered twice daily for 21 days to adult patients    with solid tumors. J Clin Oncol 15:1087-1093.-   Crinelli R, Bianchi M, Menotta M, Carloni E, Giacomini E, Pennati M,    Magnani M (2008) Ubiquitin over-expression promotes E6AP    autodegradation and reactivation of the p53/MDM2 pathway in HeLa    cells. Mol Cell Biochem 318:129-145.-   Cruz-Correa M, Zhao R, Oviedo M, Bernabe R D, Lacourt M, Cardona A,    Lopez-Enriquez R, Wexner S, Cuffari C, Hylind L, Platz E, Cui H,    Feinberg A P, Giardiello F M (2009) Temporal stability and    age-related prevalence of loss of imprinting of the insulin-like    growth factor-2 gene. Epigenetics 4:114-118.-   Dan B (2008) Angelman Syndrome London: Mac Keith Press. 256 pp.-   Dan B (2009) Angelman syndrome: current understanding and research    prospects. Epilepsia 50:2331-2339.-   Dindot S V, Antalffy B A, Bhattacharjee M B, Beaudet A L (2008) The    Angelman syndrome ubiquitin ligase localizes to the synapse and    nucleus, and maternal deficiency results in abnormal dendritic spine    morphology. Hum Mol Genet 17:111-118.-   Ehninger D, Li W, Fox K, Stryker M P, Silva A J (2008) Reversing    neurodevelopmental disorders in adults. Neuron 60:950-960.-   Elphick G F, Querbes W, Jordan J A, Gee G V, Eash S, Manley K, Dugan    A, Stanifer M, Bhatnagar A, Kroeze W K, Roth B L, Atwood W J (2004)    The human polyomavirus, JCV, uses serotonin receptors to infect    cells. Science 306:1380-1383.-   Feun L, Savaraj N (2008) Topoisomerase I inhibitors for the    treatment of brain tumors. Expert Rev Anticancer Ther 8:707-716.-   Gaulton K J, Nammo T, Pasquali L, Simon J M, Giresi P G, Fogarty M    P, Panhuis T M, Mieczkowski P, Secchi A, Bosco D, Berney T, Montanya    E, Mohlke K L, Lieb J D, Ferrer J (2010) A map of open chromatin in    human pancreatic islets. Nat Genet 42:255-259.-   Glenn C C, Driscoll D J, Yang T P, Nicholls R D (1997) Genomic    imprinting: potential function and mechanisms revealed by the    Prader-Willi and Angelman syndromes. Mol Hum Reprod 3:321-332.-   Glenn C C, Nicholls R D, Robinson W P, Saitoh S, Niikawa N, Schinzel    A, Horsthemke B, Driscoll D J (1993) Modification of 15q11-q13 DNA    methylation imprints in unique Angelman and Prader-Willi patients.    Hum Mol Genet 2:1377-1382.-   Guy J, Gan J, Selfridge J, Cobb S, Bird A (2007) Reversal of    neurological defects in a mouse model of Rett syndrome. Science    315:1143-1147.-   Hartmann J T, Lipp H P (2006) Camptothecin and podophyllotoxin    derivatives: inhibitors of topoisomerase I and II—mechanisms of    action, pharmacokinetics and toxicity profile. Drug Saf 29:209-230.-   He H H, Meyer C A, Shin H, Bailey S T, Wei G, Wang Q, Zhang Y, Xu K,    Ni M, Lupien M, Mieczkowski P, Lieb J D, Zhao K, Brown M, Liu X    S (2010) Nucleosome dynamics define transcriptional enhancers. Nat    Genet 42:343-347.-   Herben V M, Ten Bokkel Huinink W W, Beijnen J H (1996) Clinical    pharmacokinetics of topotecan. Clin Pharmacokinet 31:85-102.-   Herben V M, Ten Bokkel Huinink W W, Schellens J H, Beijnen J    H (1998) Clinical pharmacokinetics of camptothecin topoisomerase I    inhibitors. Pharm World Sci 20:161-172.-   Huang H S, Akbarian S (2007) GAD1 mRNA expression and DNA    methylation in prefrontal cortex of subjects with schizophrenia.    PLoS One 2:e809.-   Huang H S, Matevossian A, Jiang Y, Akbarian S (2006) Chromatin    immunoprecipitation in postmortem brain. J Neurosci Methods    156:284-292.-   Huang H S, Matevossian A, Whittle C, Kim S Y, Schumacher A, Baker S    P, Akbarian S (2007) Prefrontal dysfunction in schizophrenia    involves mixed-lineage leukemia 1-regulated histone methylation at    GABAergic gene promoters. J Neurosci 27:11254-11262.-   Huang X P, Setola V, Yadav P N, Allen J A, Rogan S C, Hanson B J,    Revankar C, Robers M, Doucette C, Roth B L (2009) Parallel    functional activity profiling reveals valvulopathogens are potent    5-HT2B receptor agonists: implications for drug safety assessment.    Mol Pharmacol 76:710-722.-   Jiang Y H, Armstrong D, Albrecht U, Atkins C M, Noebels J L, Eichele    G, Sweatt J D, Beaudet A L (1998) Mutation of the Angelman ubiquitin    ligase in mice causes increased cytoplasmic p53 and deficits of    contextual learning and long-term potentiation. Neuron 21:799-811.-   Keiser M J, Setola V, Irwin J J, Laggner C, Abbas A I, Hufeisen S J,    Jensen N H, Kuijer M B, Matos R C, Tran T B, Whaley R, Glennon R A,    Hert J, Thomas K L, Edwards D D, Shoichet B K, Roth B L (2009)    Predicting new molecular targets for known drugs. Nature    462:175-181.-   Kim H C, Huibregtse J M (2009) Polyubiquitination by HECT E3s and    the determinants of chain type specificity. Mol Cell Biol    29:3307-3318.-   Kim J H, Kim S H, Kolozsvary A, Khil M S (1992) Potentiation of    radiation response in human carcinoma cells in vitro and murine    fibrosarcoma in vivo by topotecan, an inhibitor of DNA    topoisomerase I. Int J Radiat Oncol Biot Phys 22:515-8.-   Kirkwood A, Dudek S M, Gold J T, Aizenman C D, Bear M F (1993)    Common forms of synaptic plasticity in the hippocampus and neocortex    in vitro. Science, 260:1518-1521.-   Koster D A, Palle K, Bot E S, Bjornsti M A, Dekker N H (2007)    Antitumour drugs impede DNA uncoiling by topoisomerase I. Nature    448:213-217.-   Kunimoto T, Nitta K, Tanaka T, Uehara N, Baba H, Takeuchi M,    Yokokura T, Sawada S, Miyasaka T, Mutai M (1987) Antitumor activity    of    7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxy-camptothecin,    a novel water-soluble derivative of camptothecin, against murine    tumors. Cancer Res 47:5944-5947.-   Liu L F, Wang J C (1987) Supercoiling of the DNA template during    transcription. Proc Nail Acad Sci USA 84:7024-7027.-   Lossie A C, Whitney M M, Amidon D, Dong H J, Chen P, Theriaque D,    Hutson A, Nicholls R D, Zori R T, Williams C A, Driscoll D J (2001)    Distinct phenotypes distinguish the molecular classes of Angelman    syndrome. J Med Genet 38:834-845.-   Miura K, Kishino T, Li E, Webber H, Dikkes P, Holmes G L, Wagstaff    J (2002) Neurobehavioral and electroencephalographic abnormalities    in Ube3a maternal-deficient mice. Neurobiol Dis 9:149-159.-   Moeschler J B, Mohandas T K, Hawk A B, Noll W W (2002) Estimate of    prevalence of proximal 15q duplication syndrome. Am J Med Genet    111:440-442.-   Morison I M, Reeve A E (1998) Insulin-like growth factor 2 and    overgrowth: molecular biology and clinical implications. Mol Med    Today 4:110-115.-   Motl S, Zhuang Y, Waters C M, Stewart C F (2006) Pharmacokinetic    considerations in the treatment of CNS tumours. Clin Phctrmacokinet    45:871-903.-   Moy S S, Nadler J J (2008) Advances in behavioral genetics: mouse    models of autism. Mol Psychiatry 13:4-26.-   Moy S S, Nadler J J, Magnuson T R, Crawley J N (2006) Mouse models    of autism spectrum disorders: the challenge for behavioral genetics.    Am J Med Genet C Semin Med Genet 142:40-51.-   Moy S S, Nadler J J, Perez A, Barbaro R P, Johns J M, Magnuson T R,    Piven J, Crawley J N (2004) Sociability and preference for social    novelty in five inbred strains: an approach to assess autistic-like    behavior in mice. Genes Brain Behav 3:287-302.-   Moy S S, Nadler J J, Young N B, Normeman R J, Grossman A W, Murphy D    L, D'Ercole A J, Crawley J N, Magnuson T R, Lauder J M (2009) Social    approach in genetically engineered mouse lines relevant to autism.    Genes Brain Behav 8:129-142.-   Moy S S, Nadler J J, Young N B, Perez A, Holloway L P, Barbaro R P,    Barbaro J R, Wilson L M, Threadgill D W, Lauder J M, Magnuson T R,    Crawley J N (2007) Mouse behavioral tasks relevant to autism:    phenotypes of 10 inbred strains. Behav Brain Res 176:4-20.-   O'Connor K A, Roth B L (2005) Finding new tricks for old drugs: an    efficient route for public-sector drug discovery. Nat Rev Drug    Discov 4:1005-1014.-   Pelc K, Cheron G, Dan B (2008) Behavior and neuropsychiatric    manifestations in Angelman syndrome. Neuropsychiatr Dis Treat    4:577-584.-   Philpot B D, Cho K K, Bear M F (2007) Obligatory Role of NR2A for    Metaplasticity in Visual Cortex. Neuron 53:495-502.-   Reik W (2007) Stability and flexibility of epigenetic gene    regulation in mammalian development. Nature 447:425-432.-   Roberts S E, Dennis N R, Browne C E, Willatt L, Woods G, Cross I,    Jacobs P A, Thomas S (2002) Characterisation of interstitial    duplications and triplications of chromosome 15q11-q13. Hum Genet    110:227-234.-   Rodriguez-Galindo C, Radomski K, Stewart C F, Furman W, Santana V M,    Houghton P J (2000a) Clinical use of topoisomerase I inhibitors in    anticancer treatment. Med Pediatr Oncol 35:385-402,-   Rodriguez-Galindo C, Poquette C A, Marina N M, Head D R, Cain A,    Meyer W H, Santana V M, Pappo A S (2000b) Hematologic abnormalities    and acute myeloid leukemia in children and adolescents administered    intensified chemotherapy for the Ewing sarcoma family of tumors. J    Pediatr Hematol Oncol 22:321-329.-   Rose P G, Markman M, Bell J G, Fusco N L (2006) Sequential prolonged    oral topotecan and prolonged oral etoposide as second-line therapy    in ovarian or peritoneal carcinoma: a phase I Gynecologic Oncology    Group study. Gynecol Oncol 102:236-239.-   Roth B L, Sheffler D J, Kroeze W K (2004) Magic shotguns versus    magic bullets: selectively non-selective drugs for mood disorders    and schizophrenia. Nat Rev Drug Discov 3:353-359.-   Sato M, Stryker M P (2010) Genomic imprinting of    experience-dependent cortical plasticity by the ubiquitin ligase    gene Ube3a. Proc Natl Acad Sci USA 107:5611-5617.-   Sawtell N B, Frenkel M Y, Philpot B D, Nakazawa K, Tonegawa S, Bear    M F (2003) NMDA receptor-dependent ocular dominance plasticity in    adult visual cortex. Neuron 38:977-985.-   Schellens J H, Creemers G J, Beijnen J H, Rosing H, de Boer-Dennert    M, McDonald M, Davies B, Verweij J (1996) Bioavailability and    pharmacokinetics of oral topotecan: a new topoisomerase I inhibitor.    Br J Cancer 73:1268-1271.-   Schroer R J, Phelan M C, Michaelis R C, Crawford E C, Skinner S A,    Cuccaro M, Simensen R J, Bishop J, Skinner C, Fender D, Stevenson R    E (1998) Autism and maternally derived aberrations of chromosome    15q. Am J Med Genet 76:327-336.-   Seiter K (2005) Toxicity of the topoisomerase I inhibitors. Expert    Opin Drug Saf 4:45-53.-   Soejima H, Wagstaff J (2005) Imprinting centers, chromatin    structure, and disease. J Cell Biochem 95:226-233.-   Souza V, Dong Y B, Zhou H S, Zacharias W, McMasters K M (2005)    SW-620 cells treated with topoisomerase I inhibitor SN-38: gene    expression profiling. J Transl Med 3:44.-   Steffenburg S, Gillberg C L, Steffenburg U, Kyllerman M (1996)    Autism in Angelman syndrome: a population-based study. Pediatr    Neurol 14:131-136.-   Stewart C F, Zamboni W C, Crom W R, Gajjar A, Heideman R L, Furman W    L, Meyer W H, Houghton P J, Pratt C B (1996) Topoisomerase I    interactive drugs in children with cancer. Invest New Drugs    14:37-47.-   Sutcliffe J S, Nakao M, Christian S, Orstavik K H, Tommerup N,    Ledbetter D H, Beaudet A L (1994) Deletions of a differentially    methylated CpG island at the SNRPN gene define a putative imprinting    control region. Nat Genet 8:52-58.-   Tilghman S M (1999) The sins of the fathers and mothers: genomic    imprinting in mammalian development. Cell 96:185-193.-   Trushina E, Du Charme J, Parisi J, McMurray C T (2006) Neurological    abnormalities in caveolin-1 knock out mice. Behav Brain Res    172:24-32.-   van Woerden G M, Harris K D, Hojjati M R, Gustin R M, Qiu S, de    Avila Freire R, Jiang Y H, Elgersma Y, Weeber E J (2007) Rescue of    neurological deficits in a mouse model for Angelman syndrome by    reduction of alphaCaMKII inhibitory phosphorylation. Nat Neurosci    10:280-282.-   Vassal G, Pondarre C, Boland I, Cappelli C, Santos A, Thomas C,    Lucchi E, Imadalou K, Pein F, Morizet J, Gouyette A (1998)    Preclinical development of camptothecin derivatives and clinical    trials in pediatric oncology. Biochimie 80:271-280.-   Wagstaff J, Chaillet J R, Lalande M (1991) The GABAA receptor beta 3    subunit gene: characterization of a human cDNA from chromosome    15q11q13 and mapping to a region of conserved synteny on mouse    chromosome 7. Genomics 11:1071-1078.-   Wang J C (2002) Cellular roles of DNA topoisomerases: a molecular    perspective. Nat Rev Mol Cell Biol 3:430-440.-   Wang X, Sun Q, McGrath S D, Mardis E R, Soloway P D, Clark A    G (2008) Transcriptome-wide identification of novel imprinted genes    in neonatal mouse brain. PLoS One 3:e3839.-   Weeber E J, Jiang Y H, Elgersma Y, Varga A W, Carrasquillo Y, Brown    S E, Christian J M, Mirnikjoo B, Silva A, Beaudet A L, Sweatt J    D (2003) Derangements of hippocampal calcium/calmodulin-dependent    protein kinase II in a mouse model for Angelman mental retardation    syndrome. J Neurosci 23:2634-2644.-   Williams C A (2005) Neurological aspects of the Angelman syndrome.    Brain Dev 27:88-94,-   Yashiro K, Riday T T, Condon K H, Roberts A C, Bernardo D R, Prakash    R, Weinberg R J, Ehlers M D, Philpot B D (2009) Ube3a is required    for experience-dependent maturation of the neocortex. Nature    Neuroscience 12:777-783.-   Zhang J H, Chung T D, Oldenburg K R (1999) A Simple Statistical    Parameter for Use in Evaluation and Validation of High Throughput    Screening Assays. J Biomol Screen 4:67-73.-   Zori R T, Hendrickson J, Woolven S, Whidden E M, Gray B, Williams C    A (1992) Angelman syndrome: clinical profile. J Child Neurol    7:270-280.

Example 16

Angelman syndrome is a severe neurodevelopmental disorder caused bydeletion or mutation of the maternal allele of the ubiquitin proteinligase E3A(Ube3a)¹⁻³. In neurons, the paternal allele of Ube3a is intactbut epigenetically silenced⁴⁻⁶, raising the possibility that Angelmansyndrome could be treated by activating this silenced allele to restorefunctional UBE3A protein^(7,8). Using an unbiased, high-content screenin primary cortical neurons from mice, twelve topoisomerase I inhibitorsand four topoisomerase II inhibitors were identified that unsilence thepaternal Ube3a allele. These drugs included topotecan, irinotecan,etoposide, and dexrazoxane (ICRF-187).

At nanomolar concentrations, topotecan upregulated catalytically activeUBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantlydown-regulated expression of the Ube3a antisense transcript thatoverlaps the paternal copy of Ube3a⁹⁻¹¹. These results suggest thattopotecan unsilences Ube3a in cis by reducing transcription of animprinted antisense RNA. When administered in vivo, topotecan unsilencedthe paternal Ube3a allele in several regions of the nervous system,including neurons in the hippocampus, neocortex, striatum, cerebellumand spinal cord. Paternal expression of Ube3a remained elevated for atleast twelve weeks after cessation of topotecan treatment, suggestingtransient topoisomerase inhibition has enduring effects on geneexpression. These findings reveal a role for topoisomerase I and IIenzymes in epigenetic gene regulation, and suggest a therapeuticstrategy for reactivating the functional but dormant allele of Ube3a inpatients with Angelman syndrome.

No effective therapies exist for Angelman syndrome (AS)—an imprintingdisorder caused by mutations or deletions in the maternal allele ofUbe3a¹⁻³. Ube3a is biallelically expressed in most tissues of the body;however, in rodents and humans, most neurons express Ube3a only from thematernally-inherited allele^(4,12-14). This unique epigenetic pattern ofregulation suggested that it might be possible to unsilence the dormantpaternal Ube3a allele in neurons^(7,8).

To test this possibility, a 384-well high-content screen was developedusing primary mouse cortical neurons from Ube3a-Yellow FluorescentProtein (Ube3a-YFP) knockin mice¹⁵, to search for drug-like moleculesthat could unsilence the paternal Ube3a-YFP allele (FIG. 13A). Thisscreen was based on the observation that the imprinting of Ube3a-YFP wasmaintained in vitro in cultured embryonic cortical neurons. Notably,Ube3a-YFP expression was undetectable (silenced) in cultured neuronswhen paternally inherited (Ube3a^(m+/pYFP)), but was expressed whenmaternally inherited (Ube3a^(mYFP/p+)) (FIG. 13B), with expressionincreasing from 4 to 10 days in vitro (DIV) (FIG. 13C). This significantdifference between maternal and paternal UBE3A-YFP protein levelsprovided a large screening window and a Z′-factor score of 0.58(determined by statistically comparing antibody-enhanced fluorescenceintensities and variations between Maternal and paternal UBE3A-YFPsignals at DIV 10), making this high-content platform suitable forunbiased screening.

To perform the screen, Ube3a^(m+/pYFP) neurons were cultured for 7 daysand then these neurons were treated with compounds (10 μM for 72 hours)from multiple small molecule libraries enriched for central nervoussystem (CNS) actives and FDA-approved drugs (FIG. 13D, FIG. 19). Intotal, 2,306 small molecules were screened in quadruplicate, and valueswere normalized to vehicle-treatment (0.2% DMSO). While methylation andother epigenetic marks are thought to control imprinting ofUbe3a^(9,16-18), surprisingly, none of the commonly used compounds thattarget the epigenome, including chromatin remodeling drugs and DNAmethyltransferase inhibitors, unsilenced the paternal Ube3a-YFP allele.A number of compounds were identified as false positives (gray compoundsin FIG. 13D) due to their intrinsic fluorescence (FIG. 17). The initialscreen identified one compound—irinotecan, an FDA-approvedcamptothecin-based topoisomerase type I inhibitor. Irinotecan lackedintrinsic fluorescence and upregulated UBE3A-YFP fluorescence (FIGS.13D-E and FIG. 18). Irinotecan (10 μM) also upregulated paternalUBE3A-YFP protein (FIG. 13F) and endogenous UBE3A protein (FIG. 13G) inneuronal cultures from Ube3a^(m+/pYFP) and Ube3a^(m−/p+) mice (AS modelmice¹³), respectively.

Many topoisomerase I inhibitors, including irinotecan and the relatedFDA-approved drug topotecan, are derived from the natural productcamptothecin (CPT)¹⁹. To explore structure activity relationships, CPTanalogs and other topoisomerase inhibitors were tested (FIG. 14A; FIGS.19-25), all of which lack inherent fluorescence (FIG. 18). Irinotecanand topotecan were found to upregulate paternal UBE3A-YFP in a dose- andtime-dependent manner in cultured neurons, with topotecan being 20× morepotent than irinotecan (FIGS. 14A-B; FIG. 26). In contrast, an inactiveanalog of CPT (lactam E-ring-CPT) that does not inhibit topoisomerases²⁰failed to unsilence the paternal Ube3a-YFP allele (FIG. 14A; FIG. 19).Ten additional topoisomerase I inhibitors unsilenced Ube3a-YFP in adose-dependent manner, including CPT analogs and structurally distinctindenoisoquinolines (Table 3 and FIGS. 19-22). In addition, structurallydistinct topoisomerase II inhibitors (etoposide, dexrazoxane, ICRF-193,and amsacrine) also unsilenced the paternal Ube3a-YFP allele (Table 3and FIGS. 23-25). The remaining studies were focused on thetopoisomerase I inhibitor topotecan because it is approved for use inhumans, it unsilenced Ube3a in the low nanomolar range, and topotecan(300 nM, 72 h) restored UBE3A protein to wild-type levels in culturedneurons from Ube3a^(m−/p+) mice (FIG. 14C).

Many topoisomerase inhibitors, including topotecan, covalently linktopoisomerases to DNA, forming stable DNA-enzyme complexes that areseparable from free topoisomerase enzymes¹⁹. Since topotecan inhibitstopoisomerase I (TOP1) and Top1 is expressed at high levels in thedeveloping and adult brain^(19,21), a subsequent analysis was focused onthis enzyme. Topotecan (300 nM, 72 h) was found to significantly reducethe amount of free TOP1 (FIG. 14D) in cultured neurons, indicating thattopotecan engages its known molecular target at doses that unsilence thepaternal Ube3a allele. These data with 16 topoisomerase inhibitors andone inactive analog strongly suggest that inhibition of topoisomerase Ior II can unsilence the paternal Ube3a allele.

UBE3A is a HECT (homology to E6 carboxyl terminus) domain E3 ligase thatforms a thioester-ubiquitin intermediate in the presence of E1 and E2enzymes²². This thioester-ubiquitin intermediate is required for HECTdomain E3 lipases to mono- and polyubiquitinate their substrates²³.Interestingly, it was noted that topotecan (300 nM, 72 hr) upregulatedUBE3A protein in Ube3a^(m−/p+) cultures along with a higher molecularweight form (resolved after running gels for longer times; FIG. 14E).This high molecular weight band was also seen in wild-type(Ube3a^(m+/p+)) cultures and was lost upon addition of the reducingagent dithiothreitol (DTT) (FIG. 14E). These data suggest that theunsilenced paternal copy of UBE3A is catalytically active and competentto form a thioester-ubiquitin intermediate, just like wild-type,maternal-derived UBE3A²³.

To further demonstrate that UBE3A was catalytically active, UBE3A wasimmunoprecipitated from cultured wild-type and Ube3a^(m−/p+) neurons(+/− topotecan), then these samples were tested for a gel-mobility-shiftin the presence or absence of the ubiquitin E2 UBCH7²⁴. Both wild-type(maternal-derived) and topotecan-unsilenced (paternal-derived) UBE3Aunderwent mobility shifts in the presence of UBCH7 plus free ubiquitinthat were abolished by DTT (FIG. 14F). This observation indicates themobility shift was due to addition of covalent ubiquitin, anddemonstrates that topotecan can upregulate a functional UBE3A enzyme.

Ube3a is repressed in cis by a large antisense transcript (Ube3a-ATS)that overlaps the paternal allele of Ube3a (FIG. 14G)^(9,10). Ube3a-ATSis expressed exclusively from the paternal allele as a result ofallele-specific methylation of an imprinting center that overlaps theUbe3a-ATS and Snurf/Snrpn transcription start site²⁵. Studies were nextcarried out to determine if topotecan regulated Ube3a expression throughchanges in Ube3a-ATS expression or imprinting center methylation.Remarkably, topotecan upregulated expression of Ube3a in culturedneurons from Ube3a^(m−/p+) mice while concomitantly downregulatingexpression of Ube3a-ATS and Snrpn (FIG. 14H). However, topotecan did notalter methylation at the imprinting center (FIG. 14I, FIG. 27). Takentogether, these data suggest that topotecan unsilences paternal Ube3a byreducing transcription of a regulatory antisense RNA without appreciablyaffecting genomic methylation at the imprinting center.

Studies were then conducted to determine if topotecan could unsilencethe paternal Ube3a allele in vivo. First, a dose was identified that waswell tolerated, meaning there were no significant decreases in bodyweight between the beginning and end of the drug treatments (FIG. 28).Topotecan (3.74 μg/h) was then administered unilaterally into thelateral ventricle of Ube3a^(m+/pYFP) or Ube3a^(m−/p+) mice byintracerebroventricular (i.c.v.) infusion for two weeks using deliveryby mini-osmotic pump, and the mice were sacrificed either immediately or5 hr after drug cessation. Strikingly, topotecan unsilenced paternalUbe3a in the hippocampus, striatum, and cerebral cortex of the infusedhemisphere, but had only a modest effect on the contralateral(non-infused) hemisphere with no effect in the cerebellum (FIGS. 15A-E,FIGS. 29-30). Pharmacokinetic analyses demonstrated that a significantamount of topotecan was detectable in the infused hemisphere immediatelyfollowing treatment whereas low levels were present in the contralateral(non-infused) hemisphere and in cerebellum (FIG. 3A, FIG. 29). However,a higher dose of topotecan (21.6 μg/h for five days) did unsilence thepaternal allele of Ube3a in Purkinje neurons of the cerebellum (FIG.31). No significant difference in topotecan levels was detected in bloodbetween drug- and vehicle-treated mice (data not shown). Topotecanconcentrations significantly declined five hours after cessation ofi.c.v. drug delivery (FIG. 15A), indicating that topotecan does notpersist and is rapidly removed/metabolized in the brain. Together, thesepharmacokinetic and pharmacodynamic data suggest that the degree towhich topotecan unsilences the paternal Ube3a allele is directlycorrelated with drug concentrations in the brain. Moreover, these dataindicate topotecan has the potential to unsilence the paternal Ube3aallele throughout the nervous system.

Genomic imprinting is thought to be established only during prescribedgermline and embryonic periods of development and imprinted genestypically remain epigenetically regulated throughout life²⁶. Thus, itwas next sought to determine if topotecan had transient or long-lastingeffects on paternal Ube3a expression. To test this possibility, anintrathecal (i.t.) delivery protocol was employed (FIG. 15F) sincetopotecan (FIG. 15G) and irinotecan (not shown) unsilenced paternalUbe3a in a sparse population of lumbar spinal neurons, allowing thequantification of all UBE3A-YFP-positive neurons. Moreover, i.t.delivery has been used to deliver topotecan to the brain in humans²⁷. Itwas found that topotecan (50 nmol/5 μL i.t. once daily, for 10 of 14days) was well tolerated (FIG. 28) and significantly increased thenumber of paternal UBE3A-YFP-positive cells in the lumbar spinal cord ofmice (FIGS. 15G-H; FIG. 32A). The vast majority (>93%) of theseUBE3A-YFP-positive cells were NeuN⁺ neurons (FIGS. 32A-B), indicatingtopotecan unsilences Ube3a primarily in neurons in vivo. Moreover, theunsilenced paternal UBE3A-YFP protein was expressed at levels comparableto maternal UBE3A-YFP controls (FIG. 32C). Remarkably, the number ofUBE3A-YFP-positive neurons remained elevated 12 weeks followingcessation of drug treatment (FIGS. 15G-H), much longer than theelimination of topotecan from tissue (FIG. 15A). These results indicatethat topotecan can enduringly unsilence paternal Ube3a in a subset ofspinal neurons and suggest that a single course of drug treatment hasthe capacity to permanently modify expression of Ube3a.

The discovery that topoisomerase inhibitors unsilence the paternal Ube3aallele in neurons represents demonstrates that a small molecule drug canunsilence a monoallelically expressed gene. Ube3a expression is modestlyupregulated in the brain of Top2b knockout mice²⁸, providing geneticsupport that topoisomerases regulate Ube3a expression. It was found thatthe unsilenced paternal allele of UBE3A is functional, suggesting thatdrug treatments could rescue molecular, cellular, and behavioraldeficits associated with loss of UBE3A^(7,13,29). Topotecan andirinotecan are approved for use in patients with cancer, arewell-tolerated when administered chronically in adult and pediatricpatients, and penetrate into the central nervous system^(27,30). Thus,this study raises the possibility that topoisomerase inhibitors could beused to treat symptoms associated with AS and suggests thattopoisomerase inhibitors regulate gene expression through atranscriptional mechanism.

Methodology Details

All animal procedures were approved by the University of North Carolinaat Chapel Hill Animal Care and Use Committee. Ube3a-YFP knockin mice¹⁵,Ube3a^(m−/p+) mice¹³ and their age-matched, wild-type controls wereused. High-content screening was performed on a BD Pathway 855 system.UBE3A-YFP was detected for drug screening with an anti-GFP antibody(Novus Biologicals, NB600-308; 1:1000) because intrinsic YFPfluorescence levels were low in cultured neurons and tissue sections.All data are presented as mean±s.e.m., with sample sizes (n) shown infigures or stated in text. Statistical analyses were performed usingSigmaPlot 11 (Systat Software). Normality tests (Shapiro-Wilk) and equalvariance tests were run and passed (P>0.05) before parametricstatistical analyses were run.

Mice.

Ube3a-YFP mice were generated and provided by the laboratory of Dr. ArtBeaudet¹⁵. Ube3a-deficient mice were generated in the laboratory of Dr.Art Beaudet¹³ and backcrossed by Dr. Yong-hui Jiang onto a C57BL/6Jbackground. C57BL/6 mice (Charles River Laboratories) were used formatings with Ube3a-YFP, and C57BL/6J mice (Jackson Laboratories) wereused for matings with Ube3a-deficient mice and CAST/EiJ mice (JacksonLaboratories).

Primary Neuron Cultures.

Embryonic day (E) 13.5 to E16.5 mouse cortices were dissected andtrypsinized with TrypLE express at 37° C. for 10 min. Dissociatedneurons were seeded onto 384-well plates coated with poly-D-lysine (0.1mg/ml) at a density of 2×10⁴ cells/well (or at a density of 1.8×10⁶cells/well for six-well plates). Neurons were cultured with Neurobasalmedium with 5% fetal bovine serum, GlutaMax (Invitrogen, #35050-061),B27 (Invitrogen, #17504-044) and Antibiotic-Antimycotic (Invitrogen,#15240-062) and changed into Neurobasal medium supplemented with 4.84μg/ml Uridine 5′-triphosphate (Sigma, U6625), 2.46 μg/ml 5 fluoro2-deoxyuridine (Sigma, F0503), GlutaMax (Invitrogen, #35050-061), B27(Invitrogen, #17504-044), and Antibiotic-Antimycotic (Invitrogen,#15240-062) at days in vitro (DIV) 1 and DIV5.

Drug Libraries and Compounds.

Multiple drug libraries were used for the screening campaign includingthe NIMH X-901 Library (source: National Institutes of Health ChemicalSynthesis and Drug Supply Program); the NIH Clinical Collection (source:National Institutes of Health), the Prestwick Library (source: PrestwickChemical), an internal Roth laboratory library comprised mainly ofcentral nervous system active small molecules (source: NationalInstitute of Mental Health Psychoactive Drug Screening Program), a smallmolecule library of DNA methyltransferase inhibitors, protein lysinemethyltransferase inhibitors and other small-molecule modulators ofepigenetic targets (source: Center for Integrative Chemical Biology andDrug Discovery, UNC-CH). Suberoylanilidehydroxamic acid (SAHA) waspurchased from Cayman Chemical. Irinotecan, zebularine, hydralazine,procainamide, 5-aza-2′-deoxycytidine (decitabine), etoposide,tenoposide, amsacrine, and ICRF-193 were all obtained fromSigma-Aldrich. Topotecan, camptothecin (CPT), 10-hydroxy-CPT,7-ethyl-CPT, and 7-ethyl-10-hydroxy-CPT (SN38) were obtained from MOLCANCorporation. ICRF-187 was provided by the National Cancer Institute'sDevelopmental Therapeutics Program. Belotecan, silatecan and rubitecanwere provided by Dr. William Zamboni (UNC Eshelman School of Pharmacy).The indenoisoquinoline derivatives NSC706744, NSC725776, and NSC724998were synthesized as described^(32,33). The inactive lactam E ring CPTanalog was synthesized as described²⁰.

High-Content Screening Microscopy and Small Molecule Screening.

Primary cortical neurons were isolated from E13.5-16.5 Ube3a-YFP mice.Screening was performed in quadruplicate at DIV7 using multiple chemicallibraries and a compound concentration of 10 μM in 0.2% DMSO vehicle.After 72 h of drug exposure, neurons were fixed with 4% paraformaldehydein PBS for 35 min, permeabilized with 0.3% Triton X-100 in PBS on icefor 30 min, and blocked by 5% goat serum with 0.1% Triton X-100 in PBSat room temperature for 1 hr. Cells were incubated with a rabbitpolyclonal antibody to GFP (1:1000, Novus Biologicals, NB600-308) atroom temperature for 1 hr and then incubated with Alexa Fluor 488 goatantibody to rabbit IgG (1:200, Invitrogen, A11008) and DAPI (1:10,000,Invitrogen, D-1306) at room temperature for 30 min. Individual wells ofimmunofluorescence-processed plates were imaged for DAPI or Alexa 488fluorescence using the BD Pathway 855 high-content imaging microscopewith a 488 excitation/515 long pass filter. Antibody-enhanced UBE3A-YFPfluorescence intensities were determined from individual neurons indrug-treated wells and normalized to neurons in wells treated with 0.2%DMSO (vehicle control). Analyses were performed with custom macros andalgorithms using NIH Image J and Arrayscan Cell Profiler softwareprograms (Thermo Scientific/Cellomics). These image analyses enabledmasking of nuclei in individual neurons and determination of UBE3A-YFPfluorescence intensities in the nuclei of individual neurons (FIG. 16).Drug-induced increases of >50% were initially binned as screening ‘hits’if (1) the increases were consistently observed across replicate wellsand (2) no apparent toxicities were observed by assessing nuclearstructure of neurons co-stained with DAPI. Effective ‘hit’ compoundswere validated in formal dose-response experiments to determine relativecompound potencies (EC₅₀) and efficacies (E_(max)).

After the initial identification of irinotecan as an active, othertopoisomerase inhibitors were screened. DIV7 primary neurons fromUbe3a^(m+/pYFP) mice were dose-treated for 72 h with topoisomerase I andII inhibitors in ten point dose-responses in full and half log molarconcentrations (1 nM to 30 μM). Neurons were fixed, processed forimmunofluorescence, and UBE3A-YFP fluorescence intensities were againdetermined by high-content imaging microscopy. The dose response resultswere analyzed by least squares sigmoidal dose-response curve fittingmodels using Graphpad Prism 5.0 (Graphpad Software, Inc.). Thecalculated EC₅₀ values (potencies) and Y-value top plateau (estimatedefficacies or E_(max)) enabled comparative analyses of the relativepotency and efficacy of the identified compounds. To control forpotential false positive ‘hit’ compounds, cortical neurons fromwild-type mice, which lack Ube3a-YFP, were also treated to determine if‘hit’ compounds exhibit inherent fluorescence.

A Z′-factor score was determined to assess the appropriateness of thescreening platform by comparing UBE3A-YFP maternal and paternalfluorescence signals at DIV10. This was done by determining the meancellular UBE3A-YFP fluorescence of >1200 neurons in quadruplicate wellsfor both genotypes which were normalized to age-matched vehicle controltreated wells. The score was calculated using the following formula: Z′factor=1−((3×(SD Maternal UBE3A-YFP+SD Paternal UBE3A-YFP))/(MeanMaternal UBE3A-YFP−Mean Paternal UBE3A-YFP); where SD is the standarddeviation.

Immunofluorescence Staining in CNS Tissues.

For immunocytochemistry in brain tissues, mice were perfused with 4% PFAin 0.1M PBS, postfixed overnight, and cryoprotected with 20% sucrose in0.1M phosphate buffer (PB), pH=7.4 for two days. 60 μm sections werecollected and permeabilized with 0.3% Triton X-100 in 0.1M PB for 30min, and blocked by 5% goat serum for 1 hr. Sections were incubated withrabbit polyclonal antibody to GFP (1:1000, Novus Biologicals, NB600-308)at 4° C. overnight and then incubated with Alexa Fluor 488 goat antibodyto rabbit IgG and DAPI for 2 hr at room temperature. Images wereacquired using a Zeiss LSM 510 and 710 confocal microscopes.

For immunocytochemistry in spinal cord, lumbosacral spinal cord(approximately L1 to S2 and inclusive of the area corresponding tointrathecal injection site) was removed from each mouse andimmersion-fixed for 8 h in cold 4% paraformaldehyde/0.1M phosphatebuffer (pH 7.4). After a period of cryoprotection in 30% sucrose in 0.1Mphosphate buffer, each spinal cord was sectioned on a cryostat at 40 μm.Sections to be stained immediately were collected in PBS; sections to besaved for future study were placed in a PBS/ethylene glycol/glycerolsolution and stored at −20° C. Every fourth section was incubatedovernight in a mixture containing a chicken IgY to GFP (1:750; AyesLabs, GFP-1020) and a mouse IgG₁ to NeuN (1:250; Millipore, MAB377) andtreated the following day with a cocktail containing goat anti-chickenIgY-Alexa 488 (1:200; Invitrogen, A-11039), goat anti-mouse IgG1-Alexa568 (1:200; Invitrogen, A-21124), and DRAQ5 (1:10,000; Axxora,BOS-889-001). Immunostained sections were mounted from PBS ontoSuperFrostPlus Slides (Fisher), which were then air-dried briefly,rehydrated with PBS, and coverslipped with FluoroGel (ElectronMicroscopy Sciences). For quantification studies, sections were imagedusing a Nikon Eclipse 80i with Surveyor mosaic imaging software. Forqualitative assessment, sections were imaged as maximal projections on aZeiss LSM 510.

Optical Intensity Measurement of UBE3A-YFP in Brain Tissue and CellCounting of UBE3A-YFP-Positive Neurons in Spinal Cord.

For optical intensity measurement in selected brain regions, sectionswere imaged using a Zeiss LSM 510 confocal microscope. With custommacros created using ImageJ software³⁴, optical intensity of UBE3A-YFPwas measured in different regions of hippocampus and striatum fromvehicle- and topotecan-treated mice. Image intensity levels werenormalized to background intensities from appropriate regions invehicle-treated mice. Brain sections between Bregma −1.22 mm and −2.06mm were chosen for analysis (n=5 sections/mouse for hippocampus and n=3to 5 sections/mouse for striatum).

For cell counting in spinal cord, 14 to 18 sections per mouse insegments corresponding to the injection site were analyzed. Slices wereimaged using a Nikon Eclipse 80i microscope equipped with aQimagingRetigaExi high-speed CCD camera system and Surveyor mosaicimaging software using a 10× objective. For qualitative purposes,selected sections were also imaged on a Zeiss LSM 510 confocalmicroscope. Cells were counted manually by individuals blind to theexperimental groups from raw (unprocessed) images using ImageJ andCellprofiler³⁵ software.

YFP intensity levels from confocal XYZ image stacks were measured usinga semi-automated macro with ImageJ. Individual YFP-positive cells wereselected by eye, the criteria being the Z plane having the largest areafor each cell. Cell regions were defined by intensity thresholding ormanual tracing and the average YFP intensity and percent saturation werecalculated for each cell.

Western Blotting.

E13.5-15.5 primary cortical neurons from Ube3a-YFP, Ube3a-deficient, orwild-type mice were plated in 6-well plates. At DIV7, neurons weretreated with drug or 0.1% DMSO for 72 h, and then total protein lysateswere obtained by lysis buffer (1% Triton X-100, 5 mM EDTA, 0.15M NaCl,10 mM Tris-HCl, pH 7.5, phosphatase inhibitor cocktails 1, proteaseinhibitor cocktail). To assess UBE3A-YFP or UBE3A protein levels, 7.5 μgof total protein lysates from Ube3a-YFP or Ube3a-deficient neurons wereseparated by 8% SDS-polyacrylamide gel electrophoresis. Proteins werethen transferred to nitrocellulose membranes, and immunoblotting wasperformed using a rabbit anti-GFP antibody (Novus, 1:500) and AlexaFluor 680 goat anti-rabbit IgG (Invitrogen, 1:5000) or rabbit anti-UBE3Aantibody (Abeam, 1:500) and Alexa Fluor 680 goat anti-rabbit IgG(Invitrogen, 1:5000). UBE3A-YFP or native UBE3A protein bands werevisualized by an Odyssey system (LI-COR Biosciences). To control forprotein loading, UBE3A-YFP or UBE3A protein levels were normalized toactin levels detected in each sample.

Ubiquitin Thioester Assay.

DIV10 cortical neurons were harvested and lysed in immunoprecipitationbuffer (20 mM Tris-HCl, 3 mM EDTA, 3 mM EGTA, 150 mM NaCl, 1% TritonX-100, pH 7.4) containing 10 mM sodium fluoride, 1.0 mM sodiumorthovanadate, 1.0 μg/mL aprotinin, and 0.1 mM DTT. UBE3A protein wasimmunoprecipitated from cell extracts with 5.0 μg of an anti-UBE3Aantibody (Bethyl Laboratories) overnight at 4° C. and then washed 2times with IP buffer containing 500 mM NaCl, followed by ubiquitinbuffer (50 mM Tris, 5 mM MgCl₂, pH 7.6). For in vitro ubiquitination ofimmunoprecipitated UBE3A, UBE3A was mixed with 0.1 μg E1, 0.5 μg UBCH7,2.5 μg ubiquitin (Boston Biochem) and 10 mM ATP in a total reactionvolume of 20 μL. The reaction was incubated at room temperature for 10min and end products were stopped in 2×SDS sample buffer with or withoutDTT. Samples were boiled, separated by SDS PAGE gel electrophoresis,transferred to PVDF membrane, and immunoblotted with an anti-UBE3A oranti-ubiquitin (Santa Cruz) antibody.

Topoisomerase Depletion Assay.

DIV7 cortical neurons were treated with 300 nM topotecan or vehicle for72 h. Cells were harvested and lysed on ice for 30 min in RIPA buffer(50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate,0.1% SDS, pH 7.4) to preserve topoisomerase cleavable complexes. Cellextracts were then centrifuged at 14,000 rpm at 4° C. to pelletinsoluble debris. 30 μg of the cell supernatant was diluted in 2×SDSsample buffer and separated on a 7.5% SDS-PAGE gel, transferred to PVDFmembrane, and immunblotted with an anti-Topoisomerase I antibody (SantaCruz) to detect free Topoisomerase I. An anti-β-Tubulin (Sigma) antibodywas used as a loading control.

Quantitative PCR.

For quantitative reverse transcriptase polymerase chain reaction(qRT-PCR) analysis, total RNA was extracted from cortical culturestreated with 300 nM topotecan using Trizol reagent (Invitrogen). Firststrand cDNA synthesis was performed on 500 ng-2 μg total RNA usingSuperscript III reverse transcriptase (Invitrogen) primed with randomhexamers. qPCR reactions used SYBR green (Invitrogen) and were run on aRotorgene 3000 (Corbett Research). Standard curves and Ct values weregenerated using Rotorgene analysis software version 6.0. Expression ofUbe3a, Ube3a-ATS, Ipw and Snrpn were determined after normalization ofeach cDNA sample to expression levels of the housekeeping genes Rpl22and Actb. Primers used were as shown in Table 4.

Bisulfite Sequencing.

Genomic DNA was extracted from cortical cultures treated with 300 nMtopotecan using Trizol reagent. Bisulfite conversion was performed on200 ng genomic DNA using the MethylCode kit (Invitrogen). 1 μL ofconverted DNA was used as template for PCR. A first round of PCR wasperformed as described in Peery et al. using the W18(5′-GTAGTAGGAATGTTTAAGTATTTTTTTTGG) and W19(5′-CCAATTCTCAAAAATAAAAATATCTAAATT) or W16(5′-AATTTAGATATTTTTATTTTTGAGAATTGG) and W17(5′-TCTACAAATCCCTACAACAACAACAA) primer sets³⁸. A second semi-nestedround of PCR was performed using 1 μL of the primary PCR reaction astemplate. For semi-nested PCR, the W18 primer was used with the nestedW19-inside primer (5′-AAATAAAATACACTTTCACTACTAAAATC), or the W16 primerwas used with the nested W 17-inside primer (5′-ACAACAAAACTTCTATCCACAC).Products were separated on an agarose gel and extracted using theQiaquick gel extraction kit (Qiagen). Purified DNA was ligated into thepGem-T Easy vector (Promega). Individual clones were amplified andsequenced.

Intrathecal Injection.

Topotecan (50 nmol in 5 unless noted) was injected into unanesthetizedmice via a 30-gauge needle attached to a 50 μL Luer-hub Hamilton syringeusing the direct lumbar puncture method³⁹; injections were made at lowerlumbar levels. Following the injection, the syringe is rotated slightlyand removed. Topotecan was dissolved with 10% DMSO or 50 mM tartrateacid buffer in 0.9% saline. Comparable vehicle injections were made incontrol mice.

Intracerebroventricular Drug Infusions.

Mice with paternal Ube3a-YFP (Ube3a^(m+/pYFP)) were anesthetized withketamine/xylazine (120 mg/kg; 9 mg/kg) and positioned in a stereotaxicapparatus. The scalp was shaved and cut, and the skull exposed. Thelocal anesthetic (bupivacaine, 2.5 mg/ml) was applied to the skull, andmineral oil was applied to protect the eyes of the mice. Acetone wasapplied on the skull to remove any lipid tissues on the skull as well asto dry the skull surface for optimal adhesion. Next, a cannula (BrainInfusion Kit 1, DURECT Corporation) was positioned into a lateralventricle at the following coordinates (−0.3 mm A/P, +1.0 mm M/L, −2.5mm D/V), as described⁴⁰. The free end of the cannula was connected to amini-osmotic pump (Alzet, Model 2001 or 2002) via a 2.5 cm piece ofpolyethylene (PE) tubing (DURECT Corporation). The mini-osmotic pump andthe connecting PE tubing were filled with, respectively for Alzet models2002 and 2001, 16.34 mM or 47.17 mM topotecan (CPT06, MolcanCorporation) dissolved in 50 mM tartaric acid with 0.9% saline, unlessindicated. The filled pumps were incubated in sterile saline at 37° C.for at least 4 hr before being implanted under the dorsal skin of themouse's back. The cannula base and the attached piece of PE tubing werefixed to the skull with Loctite cyanoacrylic 454. The incision site wasclosed with prolene suture. During and after surgery, mice were placedon a heating pad to maintain body temperature. 14 days (Alzet model2002) or five days (Alzet model 2001) following minipump implantation,mice were sacrificed following pentobarbital overdose (150 mg/kg, i.p.)and brains were removed for immunofluorescence staining orpharmacokinetic analysis.

Pharmacokinetic Analysis of Topotecan.

Total topotecan concentrations in blood and brain homogenate weredetermined by Liquid Chromatography/Triple Quadrupole Mass Spectrometry(HPLC/MS-MS). The HPLC-MS/MS system consisted of two Shimadzu Scientific(Columbia, Md.) solvent delivery pumps, a Valco (Houston, Tex.)switching valve, a thermostated (6° C.) LEAP HTC autosampler (Carrboro,N.C.), and an Applied Biosystems (Foster City, Calif.) API3000 triplequadruple mass spectrometer. Reversed-phase gradient chromatography wasused to elute the compounds from an Aquasil (C18 3 μm, 50×2.1 mm)analytical column at a flow rate of 0.3 mL/min, following a 10 μLinjection. Starting conditions for each injection were 85% aqueous (0.1%v/v formic acid in water) and 15% organic (0.1% v/v formic acid inmethanol). This was held constant for 0.7 min. After 0.7 min, theorganic phase increased linearly to 95% 4.5 min post-injection. Thesolvent composition was held at 95% organic for 0.5 min to wash thecolumn. The column was re-equilibrated to starting conditions for thefinal 1 min. Total run time was 6 min. Eluent was diverted to waste forthe first 1.5 min. After 1.5 min post-injection, 100% of the eluent wasdirected to the mass spectrometer. The mass spectrometer was connectedto the HPLC system by a TurboIonSpray interface. User controlledvoltages, gas pressures, and source temperature were optimized viadirect infusion of topotecan, d6-topotecan (internal standard) andirinotecan (internal standard). All were analyzed in positive ion modeusing the following transitions preset in multiple reaction monitoringscans: topotecan 422.0→377.1, d6-topotecan (428.0→377.1) and irinotecan587.2→587.2 (parent to parent transition). To eliminate instrument errordue to either in source fragmentation or to cross-talk, care was takento ensure analytical separation between topotecan and its desmethylmetabolite. Automated sample acquisition and data analysis was performedusing Analyst software (version 1.4.1, Applied Biosystems, Foster City,Calif.). Calibration curves, prepared from standards prepared induplicate using appropriate matrix, were generated based on peak arearatios (analyte:internal standard) and followed a linear fit with 1/x²weighting. The lower limit of topotecan quantitation (LLQ) was 0.01 μMin both blood and brain homogenate (0.03 ng/mg brain). Unknown brain andbrain homogenate samples and spiked matrix standards were analyzedfollowing addition of internal standard and protein precipitation using4× volume of methanol containing formic acid (0.1% v/v). Note that brainhomogenates were taken from entire hemispheres; because the homogenatesincluded the ventricular cerebrospinal fluid where drug is likely toaccumulate, the drug concentrations likely overestimate theconcentrations in parenchymal brain tissue.

REFERENCES FOR EXAMPLE 16

-   1. Kishino, T., Lalande, M. & Wagstaff, J. UBE3A/E6-AP mutations    cause Angelman syndrome. Nature Genetics 15, 70-73 (1997).-   2. Matsuura, T. et al. De novo truncating mutations in E6-AP    ubiquitin-protein ligase gene (UBE3A) in Angelman syndrome. Nature    Genetics 15, 74-77 (1997).-   3. Sutcliffe, J. S. et al. The E6-Ap ubiquitin-protein ligase    (UBE3A) gene is localized within a narrowed Angelman syndrome    critical region. Genome Research 7, 368-377 (1997).-   4. Albrecht, U. et al. Imprinted expression of the murine Angelman    syndrome gene, Ube3a, in hippocampal and Purkinje neurons. Nature    Genetics 17, 75-78 (1997).-   5. Rougeulle, C., Glatt, H. & Lalande, M. The Angelman syndrome    candidate gene, UBE3A/E6-AP, is imprinted in brain. Nature Genetics    17, 14-15 (1997).-   6. Vu, T. H. & Hoffman, A. R. Imprinting of the Angelman syndrome    gene, UBE3A, is restricted to brain. Nature Genetics 17, 12-13    (1997).-   7. Mabb, A. M., Judson, M. C., Zylka, M. J. & Philpot, B. D.    Angelman syndrome: insights into genomic imprinting and    neurodevelopmental phenotypes. Trends in Neurosciences 34(6):293-303    (2011).-   8. Peters, S. U. et al. Double-blind therapeutic trial in Angelman    syndrome using betaine and folic acid. American Journal of Medical    Genetics 152A, 1994-2001 (2010).-   9. Chamberlain, S. J. & Brannan, C. I. The Prader-Willi syndrome    imprinting center activates the paternally expressed murine Ube3a    antisense transcript but represses paternal Ube3a. Genomics 73,    316-322 (2001).-   10. Landers, M. et al. Regulation of the large (approximately 1000    kb) imprinted murine Ube3a antisense transcript by alternative exons    upstream of Snurf/Snrpn. Nucleic Acids Res 32, 3480-3492 (2004).-   11. Numata, K., Kohama, C., Abe, K. & Kiyosawa, H. Highly parallel    SNP genotyping reveals high-resolution landscape of mono-allelic    Ube3a expression associated with locus-wide antisense transcription.    Nucleic Acids Res 39, 2649-2657 (2011).-   12. Nakatani, J. et al. Abnormal behavior in a chromosome-engineered    mouse model for human 15q11-13 duplication seen in autism. Cell 137,    1235-1246 (2009).-   13. Jiang, Y. H. et al. Mutation of the Angelman ubiquitin ligase in    mice causes increased cytoplasmic p53 and deficits of contextual    learning and long-term potentiation. Neuron 21, 799-811 (1998).-   14. Miura, K. et al. Neurobehavioral and electroencephalographic    abnormalities in Ube3a maternal-deficient mice. Neurobiology of    Disease 9, 149-159 (2002).-   15. Dindot, S. V., Antalffy, B. A., Bhattacharjee, M. B. &    Beaudet, A. L. The Angelman syndrome ubiquitin ligase localizes to    the synapse and nucleus, and maternal deficiency results in abnormal    dendritic spine morphology. Hum. Mol. Genet. 17, 111-118 (2008).-   16. Rougeulle, C., Cardoso, C., Fontes, M., Colleaux, L. &    Lalande, M. An imprinted antisense RNA overlaps UBE3A and a second    maternally expressed transcript. Nature Genetics 19, 15-16 (1998).-   17. Runte, M. et al. The IC-SNURF-SNRPN transcript serves as a host    for multiple small nucleolar RNA species and as an antisense RNA for    UBE3A. Hum. Mol. Genet. 10, 2687-2700 (2001).-   18. Watanabe, Y. et al. Genome-wide analysis of expression modes and    DNA methylation status at sense-antisense transcript loci in mouse.    Genomics 96, 333-341 (2010).-   19. Pommier, Y. Topoisomerase I inhibitors: camptothecins and    beyond. Nature Rev. Cancer 6, 789-802 (2006).-   20. Hertzberg, R. P. et al. Modification of the hydroxy lactone ring    of camptothecin: inhibition of mammalian topoisomerase I and    biological activity. J. Med. Chem. 32, 715-720 (1989).-   21. Plaschkes, I., Silverman, F. W. & Priel, E. DNA topoisomerase I    in the mouse central nervous system: Age and sex dependence. J.    Comp. Neurol. 493, 357-369 (2005).-   22. Scheffner, M., Nuber, U. & Huibregtse, J. M. Protein    ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester    cascade. Nature 373, 81-83 (1995).-   23. Beaudenon, S., Dastur, A. & Huibregtse, J. M. Expression and    assay of HECT domain ligases. Methods Enzymol. 398, 112-125 (2005).-   24. Kumar, S., Kao, W. H. & Howley, P. M. Physical interaction    between specific E2 and Hect E3 enzymes determines functional    cooperativity. Journal of Biological Chemistry 272, 13548-13554    (1997).-   25. Bressler, J. et al. The SNRPN promoter is not required for    genomic imprinting of the Prader-Willi/Angelman domain in mice. Nat    Genet 28, 232-240 (2001).-   26. Reik, W. Stability and flexibility of epigenetic gene regulation    in mammalian development. Nature 447, 425-432 (2007).-   27. Gammon, D. C. et al. Intrathecal topotecan in adult patients    with neoplastic meningitis. Am J Health Syst Pharm 63, 2083-2086    (2006).-   28. Lyu, Y. L. et al. Role of topoisomerase IIbeta in the expression    of developmentally regulated genes. Mol. Cell. Biol. 26, 7929-7941    (2006).-   29. Greer, P. L. et al. The Angelman Syndrome protein Ube3A    regulates synapse development by ubiquitinating arc. Cell 140,    704-716 (2010).-   30. Bomgaars, L., Berg, S. L. & Blaney, S. M. The development of    camptothecin analogs in childhood cancers. Oncologist 6, 506-516    (2001).-   32. Cushman, M. et al. Synthesis of new indeno[1,2-c]isoquinolines:    cytotoxic non-camptothecin topoisomerase I inhibitors. J Med Chem    43, 3688-3698 (2000).-   33. Nagarajan, M. et al. Synthesis and evaluation of    indenoisoquinoline topoisomerase I inhibitors substituted with    nitrogen heterocycles. J Med Chem 49, 6283-6289 (2006).-   34. Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. Image Processing    with ImageJ. Biophotonics International 11, 36-42 (2004).-   35. Lamprecht, M. R., Sabatini, D. M. & Carpenter, A. E.    CellProfiler: free, versatile software for automated biological    image analysis. Biotechniques 42, 71-75 (2007).-   36. Tsai, T. F., Armstrong, D. & Beaudet, A. L. Necdin-deficient    mice do not show lethality or the obesity and infertility of    Prader-Willi syndrome. Nat Genet 22, 15-16 (1999).-   37. Landers, M. et al. Regulation of the large (approximately 1000    kb) imprinted murine Ube3a antisense transcript by alternative exons    upstream of Snurf/Snrpn. Nucleic Acids Res 32, 3480-3492 (2004).-   38. Peery, E. G., Elmore, M. D., Resnick, J. L., Brannan, C. I. &    Johnstone, K. A. A targeted deletion upstream of Snrpn does not    result in an imprinting defect. Mamm Genome 18, 255-262 (2007).-   39. Fairbanks, C. A. Spinal delivery of analgesics in experimental    models of pain and analgesia. Adv. Drug. Deliv. Rev. 55, 1007-1041    (2003).-   40. Pierce, A. A. & Xu, A. W. De novo neurogenesis in adult    hypothalamus as a compensatory mechanism to regulate energy balance.    J Neurosci 30, 723-730 (2010).-   41. Leone, S., Cornetta, T., Basso, E. & Cozzi, R. Resveratrol    induces DNA double-strand breaks through human topoisomerase II    interaction. Cancer Lett 295, 167-172 (2010).-   42. Lopez-Lazaro, M., Calderon-Montano, J. M., Burgos-Moron, E. &    Austin, C. A. Green tea constituents (−)-epigallocatechin-3-gallate    (EGCG) and gallic acid induce topoisomerase I- and topoisomerase    II-DNA complexes in cells mediated by pyrogallol-induced hydrogen    peroxide. Mutagenesis 26, 489-498 (2011).-   43. Berger, S. J., Gupta, S., Belfi, C. A., Gosky, D. M. &    Mukhtar, H. Green tea constituent (−)-epigallocatechin-3-gallate    inhibits topoisomerase I activity in human colon carcinoma cells.    Biochem Biophys Res Commun 288, 101-105 (2001).-   44. Markovits, J. et al. Inhibitory effects of the tyrosine kinase    inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res    49, 5111-5117 (1989).-   45. Snyder, R. D. & Gillies, P. J. Reduction of genistein    clastogenicity in Chinese hamster V79 cells by daidzein and other    flavonoids. Food Chem Toxicol 41, 1291-1298 (2003).-   46. Cantero, G., Campanella, C., Mateos, S. & Cortes, F.    Topoisomerase II inhibition and high yield of endoreduplication    induced by the flavonoids luteolin and quercetin. Mutagenesis 21,    321-325 (2006).-   47. Snyder, R. D. & Gillies, P. J. Evaluation of the clastogenic,    DNA intercalative, and topoisomerase II-interactive properties of    bioflavonoids in Chinese hamster V79 cells. Environ Mol Mutagen 40,    266-276 (2002).-   48. Boege, F. et al. Selected novel flavones inhibit the DNA binding    or the DNA religation step of eukaryotic topoisomerase I. J Biol    Chem 271, 2262-2270 (1996).-   49. Lopez-Lazaro, M., Willmore, E. & Austin, C. A. The dietary    flavonoids myricetin and fisetin act as dual inhibitors of DNA    topoisomerases I and II in cells. Mutat Res 696, 41-47 (2010).

TABLE 1 Multiple structurally-distinct topoisomerase inhibitorsunsilence paternal Ube3a. Fourteen topoisomerase inhibitors have beendiscovered to unsilence the paternal Ube3a allele and increase proteinexpression in cortical neurons. These include two structurally distincttopoisomerase type 1 inhibitors, the camptothecins andindenoisoquinolines. Two topoisomerase type II inhibitors have also beenshown to unsilence Ube3a. Efficacy (E_(max)) is the level of Ube3a-YFPfluorescence intensity above control cells (0.2% DMSO treated). EC₅₀ isthe half maximal effective drug concentration in nanomoles. EfficacyParent Mechanism E_(max) Compound Compound of action (fold over control)EC₅₀ (nM) Camptothecin Belotecan Topoisomerase 1 1.85 ± 0.04 18 ± 1:1(CKD602) inhibitor Camptothecin Camptothecin Topoisomerase 1 1.66 ± 0.0632 ± 1.3 (CPT) inhibitor Camptothecin 7-Ethyl- Topoisomerase 1 2.00 ±0.09 33 ± 1.3 10-Hydroxy-CPT inhibitor Camptothecin 10-Hydroxy-CPTTopoisomerase 1 1.68 ± 0.06 40 ± 1.4 inhibitor Camptothecin RubitecanTopoisomerase 1 2.07 ± 0.08 43 ± 9.1 (9-Nitro-CPT) inhibitorCamptothecin 7-Ethyl-CPT Topoisomerase 1 1.68 ± 0.05 49 ± 1.3 inhibitorCamptothecin Topotecan Topoisomerase 1 2.25 ± 0.07 54 ± 1.2 inhibitorCamptothecin Irinotecan Topoisomerase 1 2.19 ± 0.06 994 ± 10   inhibitorCamptothecin Silatecan (DB67) Topoisomerase 1 1.71 ± 0.06 2640 ± 95  inhibitor Indenoisoquinoline NSC706744 Topoisomerase 1 1.76 ± 0.03 6.2 ±2  inhibitor Indenoisoquinoline NSC725776 Topoisomerase 1 1.82 ± 0.059.2 ± 2  inhibitor Indenoisoquinoline NSC724998 Topoisomerase 1 1.65 ±0.03 12 ± 4   inhibitor Acridine derivative Amsacrine Topoisomerase 21.65 ± 0.05 28 ± 10  inhibitor Bisdioxopiperazine ICRF-193 Topoisomerase2 2.51 ± 0.1  131 ± 13   inhibitor

TABLE 2 Lentivirus shRNA Gene NM ID Clone ID Sequence TopI NM_009408TRCN0000011883 CCGGCCACAAGTCTTAACAAACCAACTCGAGTTGGTTTGTTAAGACTTGTGGTTTTTTRCN0000011884 CCGGCCAGCGAAGATTCTATCTTATCTCGAGATAAGATAGAATCTTCGCTGGTTTTTTRCN0000011885 CCGGCGATTGAATGATTCTCACAAACTCGAGTTTGTGAGAATCATTCAATCGTTTTTTRCN0000011886 CCGGCCGCCACGAATTAAAGATGAACTCGAGTTCATCTTTAATTCGTGGCGGTTTTTTRCN0000011887 CCGGGCAGTCTAAGATTGATGCCAACTCGAGTTGGCATCAATCTTAGACTGCTTTTTTopIIa NM_011623 TRCN0000070983CCGGCCCGAGTTTGAAGAATGGAAACTCGAGTTTCCATTCTTCAAACTCGGGTTTTT TRCN0000070984CCGGCCTCTCTAATAACAGACTATACTCGAGTATAGTCTGTTATTAGAGAGGTTTTT

TRCN0000070985 CCGGGCTCGCTTTATATTAGAGAAACTCGAGTTTCTCTAATATAAAGCGAGCTTTTT

TRCN0000070986 CCGGGCAGACTACATTGCCGTTTAACTCGAGTTAAACGGCAATGTAGTCTGCTTTTTTRCN0000070987 CCGGCCAGCAGATTAGCTTCGTCAACTCGAGTTGACGAAGCTAATCTGCTGGTTTTTTopIIb NM_009409 TRCN0000070988CCGGCCTTGTGTTGTCCTTTGTCTTCTCGAGAAGACAAAGGACAACACAAGGTTTTT TRCN0000070989CCGGCCGCCAAATCTCTAGCTGTATCTCGAGATACAGCTAGAGATTTGGCGGTTTTT TRCN0000070990CCGGCGCAGCTATGTAGACCTTTATCTCGAGATAAAGGTCTACATAGCTGCGTTTTT TRCN0000070991CCGGGCTAGAGAAATTGTGAACAATCTCGAGATTGTTCACAATTTCTCTAGCTTTTT TRCN0000070992CCGGCCCATTGTAAAGGCAAGCAAACTCGAGTTTGCTTGCCITTACAATGGGTTTTT

siRNA TopI CCTTTGAGAAGTCAATGATTT CCGAAATCAGTATCGGGAATTCAATTGAGAAGATTTACAATT TopIIb CTGTTAGTGGTGAGATATTTT GACTATAAACTCTGACTCATTCCTGATACCACAGTAGTGATT

indicates data missing or illegible when filed

TABLE 3 Efficacies and potencies of topoisomerase inhibitors forunsilencing the paternal allele of Ube3a-YFP in cultured neurons.Potency Efficacy E_(max) Compound EC₂₀ (nM) (fold over vehicle)7-Ethyl-Camptothecin 7.2 ± 2.3  1.70 ± 0.04 (7-Ethyl-CPT)7-Ethyl-10-Hydroxy-CPT 11 ± 3.2 1.99 ± 0.06 10-Hydroxy-CPT 14 ± 5.71.82 + 0.08 Belotecan (CKD602) 19 ± 4.4 1.88 ± 0.05 Camptothecin (CPT)21 ± 3.8 2.11 ± 0.05 Topotecan* 54 ± 3.4 2.25 ± 0.05 Rubitecan(9-Nitro-CPT) 62 ± 18  2.09 ± 0.09 Irinotecan* 994 ± 13   2.17 ± 0.05Silatecan (DB67) 2244 ± 171   1.65 ± 0.05 Lactam E ring-CPT (inactive)inactive inactive NSC725776 10 ± 1.6 1.76 ± 0.03 NSC706744 11 ± 3.21.84 + 0.07 NSC724998 14 ± 2.2 1.69 ± 0.03 Etoposide* 1600 ± 980   1.68± 0.04 ICRF-193 205 ± 70   2.21 ± 0.09 Dexrazoxane (ICRF-187)* 20470 ±1450   1.82 ± 0.05 Amsacrine 27 ± 5.2 1.74 ± 0.06 (NSC725776, NSC706744and NSC724998 = indenoisoquinoline derivatives, Etoposide =podophyllotoxin derivative, ICRF-193 and Dexrazoxane (ICRF-187) =bis-dioxopiperazine derivatives, and Amsacrine = aminoacridinederivative; all others = camtothecin derivatives).

TABLE 4 Reference Primer Sequence (5′ to 3′) (if applicable) Ube3a FCAAAAGGTGCATCTAACAACTCA Ube3ce R GGGGAATAATCCTCACTCTCTC Snrpn 1-3 FTTGGTTCTGAGGAGTGATTTGC 36 Snrpn 3 R CCTTGAATTCCACCACCTTG 36 Ipw B/C FTCACCACAACACTGGACAAAA 37 Ipw B/C R TGCTGCTACACAGGAAAGAGG 37 Ube3a-ATS FGGCACCCTTGTTTGAAACTT Ube3a-ATS R GCTCATGACCCTGTCCTTTC Rpl22 FAAGAAGCAGGTTTTGAAG Rpl22 R TGAAGTGACAGTGATCTTG Actb FCAGCTTCTTTGCAGCTCCTT Actb R CACGATGGAGGGGAATACAG

1. A method of inducing expression of Ube3a in a cell, comprisingcontacting the cell with an effective amount of a topoisomeraseinhibitor, thereby inducing expression of Ube3a in the cell.
 2. Themethod of claim 1, wherein the cell is selected from the groupconsisting of a central neuron, a peripheral neuron, a neurondifferentiated from stem cells, a glial cell, an astrocyte, anoligodendrocyte, a microglial cell, and any combination thereof.
 3. Themethod of claim 1, wherein the cell is in a human subject and the Ube3ais paternal Ube3a.
 4. A method of treating a genomic imprinting disorderin a subject, comprising administering to the subject an effectiveamount of a topoisomerase inhibitor, thereby treating the genomicimprinting disorder in the subject.
 5. The method of claim 4, whereinthe genomic imprinting disorder is Angelman syndrome.
 6. The method ofclaim 4, wherein the genomic imprinting disorder is selected from thegroup consisting of Prader Willi syndrome, Beckwith-Wiedemann syndrome,Russell-Silver syndrome, Albright hereditary osteodystrophy, Turner'ssyndrome and any combination thereof.
 7. A method of treating a disorderassociated with an epigenetic modification in a subject, comprisingadministering to the subject an effective amount of a topoisomeraseinhibitor, thereby treating the disorder associated with the epigeneticmodification in the subject.
 8. The method of claim 7, wherein thedisorder associated with an epigenetic modification is selected from thegroup consisting of an autism spectrum disorder, depression,schizophrenia, Rett syndrome, Fragile X syndrome, and any combinationthereof.
 9. The method of claim 4, wherein the subject is a human. 10.The method of claim 1, wherein the topoisomerase inhibitor is atopoisomerase I inhibitor.
 11. The method of claim 10, wherein thetopoisomerase I inhibitor is a camptothecin derivative.
 12. The methodof claim 11, wherein the camptothecin derivative is selected from thegroup consisting of Belotecan (CKD602), Camptothecin,7-Ethyl-10-Hydroxy-CPT, 10-Hydroxy-CPT, Rubitecan (9-Nitro-CPT),7-Ethyl-CPT, Topotecan, Irinotecan, Silatecan (DB67) and any combinationthereof.
 13. The method of claim 10, wherein the topoisomerase Iinhibitor is an indenoisoquinoline derivative.
 14. The method of claim13, wherein the indenoisoquinoline derivative is selected from the groupconsisting of NSC706744, NSC725776, NSC724998 and any combinationthereof.
 15. The method of claim 1, wherein the topoisomerase inhibitoris a topoisomerase II inhibitor.
 16. The method of claim 15, wherein thetopoisomerase II inhibitor is selected from the group consisting of anacridine derivative, a bisdioxopiperazine derivative, a podophyllotoxinderivative and any combination thereof.
 17. The method of claim 16,wherein the acridine derivative is Amsacrine.
 18. The method of claim16, wherein the bisdioxopiperazine derivative is ICRF-193, dexrazoxane(ICRF-187) or a combination thereof.
 19. The method of claim 16, whereinthe podophyllotoxin derivative is etoposide.
 20. The method of claim 1,wherein the topoisomerase inhibitor is an interfering RNA molecule thattargets topoisomerase I.
 21. The method of claim 1, wherein thetopoisomerase inhibitor is an interfering RNA molecule that targetstopoisomerase II.
 22. The method of claim 1, wherein the topoisomeraseinhibitor has an efficiency E_(max) of at least 1.5 fold over control.23. The method of claim 1, wherein the topoisomerase inhibitor has anefficiency E_(max) of at least 2.5 fold over control.